ALCOHOL ADMINISTRATION ON SERUM LIPID PROFILE TOTAL PROTEIN AND LIVER ENZYMES IN RATS

ABSTRACT

The efficacy of Uvaria chamae plant species in herbal remedies may have come as a result of trial  and  error.  This  could  be  as  a  result  of  poor  information  on  the  phytochemistry, antioxidant and toxicity of this plant parts. The present study compares the in vitro and in vivo antioxidant potentials and toxicities of methanol extracts of Uvaria chamae leaves and roots. Results of in vitro antioxidant potentials revealed that the methanol extract of Uvaria chamae leaves contains vitamin A (4871±79.21 I.U) and vitamin C (1.72±0.02%) while the root extract contains vitamin A (673.28±0.00I.U) and vitamin C (1.66±0.01%). Both extracts had  equal  contents  of  vitamin  E  (8.83±0.04  mg/100g).  The  leaf  extract  scavenged  1,1- diphenyl-2-picrylhydrazyl  radical  (DPPH)  in a concentration  dependent  manner  with the correlation coefficient (R2) of 0.839 and effective concentration (EC50) of 31.19 µg/ml, while the root extract scavenged DPPH with R2, 0.778 and EC50 , 14.00 µg/ml. These results were compared  to the EC50  of ascorbic acid standard (25.29 µg/ml). The leaf and root  extracts scavenged superoxide radical in a concentration dependent manner with EC50 of 5.93 µg/ml and 719.45 µg/ml, respectively,  compared to the EC50 of ascorbic standard  (30.27 µg/ml). Both the leaf and root extracts  scavenged  hydroxyl  radical  in a  concentration  dependent manner with EC50 of 107.89 µg/ml and 912.01 µg/ml, respectively, compared to the EC50 of vitamin E standard (106.66µg/ml). The result of the study revealed that the 1000 µg/ml root extract scavenged nitric oxide radical more than the leaf extract and vitamin E standard at the same concentration. At 500 µg/ml, the  leaf extract was more effective at scavenging nitric oxide radical compared to the root extract and vitamin E standard. The leaf extract showed significantly higher (p<0.05) anti radical power (ARP) of superoxide (0.17) compared to the root extract (0.0014). However, the root extract showed significantly higher (p<0.05) ARP of DPPH (0.071) compared to the leaf extract (0.032). For the in vivo study, adult albino rats were divided into two sets (leaf and root extracts) of four groups each. Each group contained 8  rats.  Comparative  in  vivo  effects  of  the  leaf  and  root  extracts  were  determined  by investigating  the  following  parameters:  catalase  activity,  liver  marker  enzymes  (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alanine phosphatase (ALP), serum urea, serum creatinine, serum electrolytes (Na+, K+, and Cl-) and some haematological

parameters  – haemoglobin  (Hb), packed  cell volume  (PCV)  and white blood cell  (WBC)

count. Because the median lethal dose (LD50) investigation revealed one death at the dose of

5000 mg/kg b.w for the root extract and none for the leaf extract, both extracts were orally

administered at 100, 200 and 400 mg/kg b.w. In each set (i.e leaf and root extracts), group 1

received  normal saline  and served as the control while groups 2, 3, and 4  received 100, 200 and 400 mg/kg b.w doses of the extracts, respectively. At day 7 post treatment, ALP, Cl- ,K+, urea, creatinine , AST and WBC count were significantly higher (p< 0.05)   in both sets of treatment groups compared to the control. Serum Na+, Hb and PCV were significantly lower (p< 0.05) in the treatment groups compared to the control. While  the leaf extract showed significantly higher (p< 0.05) ALT activity, the root extract showed no significant difference (p>0.05). At day 14, both extracts had significantly higher (p< 0.05) catalase activity, urea, creatinine, Cl-, Na+  and WBC count.   While,  the  leaf extract had significantly higher (p< 0.05) ALT and K+, the root extract had significantly higher (p< 0.05)   AST activity when compared to the control. At day 21, the root extract showed significantly higher (p< 0.05) ALT, AST, catalase , Cl- and Hb while the groups were not significantly (p>0.05) affected by the leaf extract when compared to the control    . However, at day 28, both extracts showed significantly higher (p>0.05) ALT activity. While the root extract showed significantly higher (p< 0.05) Na+ and ALP activity, the leaf extract showed none for them when compared to the control. Histological analysis showed some levels of toxicity at doses of 100, 200 and 400 mg/kg b. w at chronic  stage (beyond  14 days  of extracts’  administration).  These results suggest that fluctuations at the initial period were as a result of the homeostatic processes in attempt for the organism  to maintain normal body functioning  at the end of the  28- day administrations   of  both  extracts.  Although  the  leaf  extract  was  more   efficacious  in maintaining  the normal body metabolism;  the moderate  toxicity  exhibited  by the extracts from LD50, ALT, AST and histopathological tests could compromise its efficacy in chronic phase of treatment.

CHAPTER ONE

INTRODUCTION

Over  the  years,  man  has  been  facing  with  the  challenges  of  preventing  and eliminating diseases in the body. The discovery of the efficacy of certain plant  species in herbal remedies by man, might have come as a result of trial and error. This however, has created some gaps in common beliefs  on the treatment of ailments among some related and unrelated human societies of the world. Phytochemical analysis on certain plant species by modern   practitioners   have   shown   some   corresponding   results   with   already   existing tradomedical information while in some cases, has differed completely thereby causing doubt in herbal treatment (Nwachukwu et al., 2011). In recent times, some plants including Uvaria chamae,  have been used as herbal  medicines  due to the presence  of phytochemicals  and antioxidants  in them  (Riby et  al., 2006).  These  Antioxidants  are  vital substances  which protect the body from damages caused by free radical-induced oxidative stress (Awah et al.,

2010). However, the herb can display some toxicological properties. The assay of enzyme activities in the body fluid of any model in question, aids the diagnosis of  the damages on the vital organs and as well, assists in the determination of its toxicity (Ajiboye et al., 2010).

1.1      Profile of Uvaria chamae

Uvaria chamae  belongs to the family of Anonacaea. It is a climbing large shrub or small tree native to the tropical rain forest of West and Central Africa where it grows as wet and coastal shrub (Okwu and Iroabuchi, 2004). It is also known as finger root or bush banana (Omajali et al., 2011). This common name refers to the fruit growing in its small  branches; the fruit carpels are in finger-like clusters, the shape giving rise to the many native names translated as bush banana, implying wildness (Irving,1961). It is commonly called by the Igala people of the eastern part of Kogi State, Nigeria as Awuloko or Ayiloko by others, Kas Kaifi by the Hausas, Mmimi Ohea/Udagu  by the Igbos, Oko Oja by the Yorubas, Akotompo by the Fula- Fante people of Ghana, Boelemimbo by the Fula-Pwaar people of Guinea Bissau, Liasa by the Yoruba- Ife people of Togo (Oliver, 2010).  It is an evergreen plant that grows about 3.6 to 4.5m  high,  cultivated  as well as  wild.  The plant  is extensively branched  with  sweet, aromatic and alternate leaves commonly used to cure diseases and heal injuries (Omajali et al., 2011)

Uvaria  chamae  in Nigeria  has a wide spread  reputation  as a medicinal plant.  The  root- decoction is used as a purgative and also as a lotion. Sap from the root and stem is applied to wounds and sores; the root is made into a drink and a body wash for oedematous condition. The root bark yields an oleo- resin that is taken internally for  catarrhal  inflammation  of mucous membranes, respiratory catarrh and gonorrhea while the root extract is used in phyto medicine for the treatment of piles ,epitasis, haematuria and haemolysis (Oliver, 2010). It is a medicinal plant used in the treatment of fever and injuries (Bukill, 1989). There are other oral claims that the plant can cure abdominal pain, used as treatment for piles, wounds, sore throat

,diarrhea etc (Bukill, 1989). In Ghana, the root with Guinea grains is used in application to the  fontanelle  for  cerebral  diseases.  Among  the  Fulai people  of  Senegal,  the root  has  a reputation as the “medicine of riches” and is taken for conditions of lassitude and senescence. It  is  also  considered  to  be  a  woman’s  medicine  used  for  amenorrhea  and  to  prevent miscarriage  and  in  Togo,  a  root-decoction  is  given  for  pains  of  childbirth  (Okwu  and Iroabuchi, 2004). It is used for the treatment of jaundice in Ivory- coast. In Sierra Leone, the root is reputed for having purgative and febrifugal properties. In Nigeria however, the root- bark is used for the treatment  of  bronchitis,  and gonorrhea  in addition  to its being used internally for catarrhal  inflammation  of mucous membranes  (Okwu and Iroabuchi,  2004).

1.2       Phytochemistry

The medicinal value of some medicinal plants has a link with the phytochemicals in them. These phytochemicals are chemical compounds that occur naturally in plants (phyto means “plant” in Greek). Some are responsible for color, smell etc. The term is generally used to refer to those chemicals that may have biological significance.  There  may be as many as

4,000  different  types.    Example  of  such  phytochemicals  include:  alkaloids,  flavonoids,

saponin, tannins etc (Palermo et al., 2014).

1.2.1    Alkaloids

Alkaloids are a group of naturally occurring chemical  compounds  (natural products)  that contain mostly basic nitrogen atoms. This group also includes some related compounds with neutral and even weakly acidic properties (Manske,  1965). Some  synthetic compounds of similar structure  are also termed  alkaloids.  In addition to  carbon,  hydrogen  and nitrogen, alkaloids may also contain oxygen, sulfur and more rarely other elements such as chlorine, bromine, and phosphorus (Manske, 1965).

Alkaloids are produced by a large variety of organisms including bacteria, fungi, plants, and animals. They can be purified from crude extracts of these organisms by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects and are used as medications, as recreational drugs, or in entheogenic rituals. Examples are the local anesthetic and stimulant cocaine, the psychedelic  psilocin, the  stimulant caffeine, nicotine, the analgesic morphine (Raymond  et al., 2010), the  antibacterial berberine,  the anticancer compound   vincristine,   the   anti   hypertension    agent,   reserpine,   the   cholinomimetic galantamine,   the  anticholinergic   agent,   atropine,   the  vasodilator   vincamine,   the   anti arrhythmia compound quinidine, the anti asthma therapeutic ephedrine, and the anti malarial drug quinine. Although,  alkaloids  act on a diversity of metabolic  systems in humans and other animals, they almost uniformly invoke a bitter taste (Rhoades, 1979).

The boundary between  alkaloids  and other nitrogen-containing  natural compounds  is  not clear-cut. Compounds like amino acid peptides, proteins, nucleotides, nucleic acid, amines, and antibiotics are usually not called alkaloids (Raj, 2004). Natural compounds containing nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually attributed

to amines rather than alkaloids. Some authors, however, consider alkaloids a special case of amines (Raj, 2004).

1.2.2    Flavonoids

Flavonoids (or bioflavonoids) (from the Latin word flavus meaning yellow that is, their color in  nature)  are  a class  of  plant  secondary  metabolites.  They were  referred  as  vitamin  P (probably because of the effect they had on the permeability of vascular capillaries) from the mid-1930s to early 50s, but the term has since fallen out of use (Mobh, 1938) .Flavonoids have hydroxyl group (OH). The effect of the hydroxyl moiety of flavonoids on protein targets varies depending on the position and number of the moiety on the flavonoid skeleton (Mobh,1938)

1.2.3    Tannin

A tannin (also known as vegetable tannin, natural organic tannins or sometimes tannoid, i.e. a type of biomolecule, as opposed to modern synthetic tannin) is an astringent,  bitter plant polyphenolic  compound  that binds to and precipitates  proteins  and various  other organic compounds   including   amino   acids   and   alkaloids.   They   form   complexes   also   with carbohydrates, bacterial cell membranes and enzymes involved in protein and carbohydrate digestion.  The  tannin  phenolic  group  is  an  excelent  hydrogen  donor  that  forms  strong hydrogen bonds with the protein’s carboxyl group (Amorati and Valgimigli, 2012). The anti carcinogenic  and anti mutagenic potentials of  tannins may be related to their anti oxidant property    (Amorati  and  Valgimigli,  2012).  The  anti-microbial  properties  seemed  to  be associated with the hydrolysis of ester linkage between gallic acid and polyols hydrolyzed after ripening of many edible fruits (Amorati and Valgimigli, 2012).

1.2.4    Total Phenolics

In  organic  chemistry,   phenols,  sometimes   called  phenolics,   are  a  class  of   chemical compounds   consisting  of  a  hydroxyl   group  (—OH)  bonded  directly  to   an  aromatic hydrocarbon group. The simplest of the class is phenol, which is also called carbolic  acid C6H5OH. Phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule (Amorati, and Valgimigli, 2012). Fig. 3 shows the

structure of total phenol.Phenolic  compounds  are  synthesized  industrially;  they  also  are  produced  by plants  and microorganisms,  with variation between and within species (Hättenschwiler  and  Vitousek,

2000). Although, similar to alcohols, phenols have unique properties and are not classified as alcohols (since the hydroxyl group is not bonded to a saturated  carbon atom). They  have higher acidities due to the aromatic ring’s tight coupling with the oxygen and a  relatively loose bond between the oxygen and hydrogen. The acidity of the hydroxyl group in phenols is commonly intermediate between that of aliphatic alcohols and carboxylic acids (their pKa is usually between 10 and 12).

Loss  of  a  positive  hydrogen  ion  (H+)  from  the  hydroxyl  group  of  a  phenol  forms  a corresponding  negative phenolate ion or phenoxide ion, and the corresponding salts which are called phenolates or phenoxides. As they are present in food consumed in human diets and in plants used in traditional medicine of several cultures, their role in human health and disease is a subject of research (Mishra and Tiwari, 2011).Some phenols are germicidal and are  used  in  formulating  disinfectants.  Others  possess  estrogenic  or  endocrine  disrupting activities.  Typical  phenolics  that  possess  antioxidant activity  have  been  characterized  as phenolic  acids  and  flavonoids  (Mishra  and  Tiwari,  2011).  Antioxidant  activity  of  plant extracts  is not  limited  to  phenolics.  Activity may also  come from the presence  of other antioxidant secondary metabolites, such as volatile oils, carotenoids and vitamins A,C and E.

are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby,  improve  the quality and  nutritional  value of food.  In plants,  oils  are basically monophenolics such as tocopherols, water-soluble polyphenols are more typical in water-soluble products like fruits, vegetables, tea, coffee, wine, among others (Mishra and Tiwari, 2011). Polyphenolic compounds are known to have antioxidant activity. This activity is due to their redox properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet  oxygen, or decomposing peroxides (Mishra and Tiwari, 2011).

1.3      Acute toxicity

Acute toxicity describes the adverse effects of a substances that result either from a single exposure or from multiple exposures in a short space of time (less than 24 hours ). Most acute toxicity data come  from animal testing  or  in vitro  testing  methods  (Walum,  1998).  The median lethal dose (LD50) is the dose required to kill half the members of a tested population after a specified   test duration (Lorke, 1983). Investigation of the acute toxicity is the first step in the toxicological  investigations  of  an unknown substance.  The index of the acute toxicity  is  the  LD50  (Lorke,  1983).  Scientific  investigation  of previously  unknown  and known plants is necessary not only because of the need to discover new drugs but to assess the toxicity faced by the users. Besides, it is important that traditionally claimed therapeutic properties  of plants be  confirmed  and its toxicity limit determined  (Prohp  amd Onoagbe, 2012).

1.4      Reactive oxygen species (ROS)

These are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides.  Reactive oxygen species are formed  as a natural byproduct of the  normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of environmental  stress (e.g. UV or heat  exposure,  ROS levels can increase dramatically  (Devasagayam  et al., 2004).   This  may result  in significant  damage  to cell structures. Cumulatively, this is known as oxidative stress. Reactive oxygen species are also generated   by  exogenous   sources   such  as  ionizing  radiation.   Normally,   cells  defend themselves against  ROS damage with enzymes such as a superoxide dismutases, catalases, lactoperoxidases,  glutathione  peroxidases  and peroxiredoxins.  Small molecule antioxidants such as ascorbic acid (vitamin C), tocopherol (vitamin E) and glutathione also play important

roles  as  cellular  antioxidants.   In  a  similar  manner,  polyphenol  antioxidants  assist   in preventing ROS damage by scavenging free radicals. In contrast, the antioxidant ability of the extracellular  space is less, the most important plasma antioxidant  in humans is  uric acid. Effects of ROS on cell metabolism are well documented in a variety of species. These include not only roles in apoptosis  (programmed  cell death) but also positive  effects such as the induction of host defence  genes and mobilisation of ion transport systems (Rada and Leto,

2008). This implicates them in control of cellular function. In particular, platelets involved in wound repair and blood homeostasis release ROS to recruit additional platelets to sites of injury.  These  also  provide  a link  to  the  adaptive  immune  system  via the recruitment  of leukocytes.  Reactive  oxygen  species  are  implicated  in  cellular  activity  to  a  variety  of inflammatory  responses  including  cardiovascular  disease.  They may also  be  involved  in hearing impairment via cochlea damage induced by elevated sound levels, in ototoxicity of drugs such as cisplatin, and in congenital deafness in both animals and humans. In general, harmful effects of reactive oxygen species on the cell are most often :

    damage of DNA

    oxidations of polyunsaturated fatty acids in lipids (lipid peroxidation)

    oxidations of amino acids in proteins

    oxidatively inactivate specific enzymes by oxidation of co-factors

1.4.1    Pathogen response

When a plant recognizes an attacking pathogen, one of the first induced reactions is to rapidly produce  superoxide (O2−) or hydrogen  peroxide (H2O2)  to  strengthen  the  cell  wall.  This prevents the spread of the pathogen to other parts of the plant, essentially  forming a net around the pathogen to restrict movement and reproduction. In the mammalian host, ROS is

induced as an antimicrobial defense. To highlight the importance of this defense, individuals with chronic granulomatous  disease who have deficiencies  in generating  ROS, are highly susceptible  to  infection  by  a  broad  range  of  microbes  including  Salmonella  enterica, Staphylococcus aureus, Serratia marcescens, and Aspergillus species (Patel et al., 1999).

1.4.2    Oxidative damage

In aerobic  organisms  the  energy  needed  to  fuel  biological  functions  is produced  in  the mitochondria via the electron transport chain. In addition to energy, reactive oxygen species (ROS) with the potential to cause cellular damage are produced. Reactive oxygen species can damage DNA, RNA, and proteins, which, in theory, contributes to the physiology of ageing. (Patel et al,1999). Reactive  oxygen  species  are  produced  as  a  normal  product  of  cellular  metabolism.  In particular, one major contributor to oxidative damage is hydrogen peroxide (H2O2), which is converted  from  superoxide  that  leaks  from  the  mitochondria.  Catalase  and  superoxide dismutase   ameliorate   the   damaging   effects   of   hydrogen   peroxide   and   superoxide, respectively, by converting these compounds into oxygen and  hydrogen peroxide (which is later converted  to water), resulting in the production of  benign molecules.  However,  this conversion is not 100% efficient, and residual peroxides persist in the cell. While ROS are produced as products of normal cellular functioning, excessive amounts can cause deleterious effects  (Patel  et  al.,  1999).  Memory  capabilities  decline  with  age,  evident  in  human degenerative diseases such as Alzheimer’s disease, which is accompanied by an accumulation of oxidative damage. Current studies demonstrate that the accumulation of ROS can decrease an organism’s fitness because oxidative damage is a contributor to senescence. In particular, the accumulation of oxidative damage may lead to cognitive dysfunction, as demonstrated in a study in which old rats were given mitochondrial metabolites and then given cognitive tests. Results showed that the rats performed better after receiving the metabolites, suggesting that the metabolites reduced oxidative damage and improved mitochondrial function (Liu et al.,2002).Accumulating  oxidative  damage can then affect the efficiency of mitochondria  and further increase the rate of ROS production (Stadtman, 1992). The accumulation of oxidative damage and its implications for aging depends on the particular tissue type where the damage is occurring. Additional experimental results suggest that oxidative damage is responsible for age-related decline in brain functioning. Older gerbils were  found to have higher levels of oxidized protein in comparison to younger gerbils. Treatment of old and young mice with a spin trapping compound caused a decrease in the level of oxidized proteins in older gerbils but did not have an effect on younger gerbils. In addition, older gerbils performed cognitive tasks better during treatment but ceased functional capacity when treatment was discontinued, causing oxidized protein levels to increase. This led  researchers to conclude that oxidation of cellular proteins is potentially important for brain function (Carney et al., 1991).

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