A COMPARISON BETWEEN THE HEPATOPROTECTIVE POTENTIALS

OF ADEDIRONIC AND IDOGUN HERBAL MIXTURES

1. ADEDIRAN O A1. And OPEYEMI O J.2

2. Department of Medical Laboratory Science.

3. Histopathology Department Federal Medical Center Owo.

Corresponding Author. ADEDIRAN O A

ABSTRACT

This study was carried out to investigate and compare the hepatoprotective

potentials of two anti-ulcer herbal mixtures both at lower and higher doses. The

two herbal mixtures were administered to 85 rats of either sex weighing between

150 and 200g .The lower concentration of the two herbal mixtures were

administered to rats in 6 groups of 5 rats for 14 days. The groups are control

100mg/kg, 400mg/kg and reference drug, 20mg Omeprazole. The higher

concentrations of the two herbal mixtures were administered to rats in four groups

(control), 500mg/kg, 1000mg/2500mg/kg for 28days. It was found that there was

hepatocellular protection at lower concentrations of the two anti-ulcer herbal

mixtures while hepatocellular degeneration was observed at higher doses of

1000mg/kg and 2500mg/kg of the two herbal extracts.

The values of aspartate aminotransferase (AST) and alanine aminotransferase

(ALT) in rats treated with the herbal mixtures at higher doses showed no

significant differences from the control values. In conclusion morphologically,

there was hepatocellular protective potentials in the two extracts especially at

lower concentrations.

Keywords; Hepatoprotective, Omeprazole, aspartate aminotransferase, alanine

aminotransferase

INTRODUCTION

Peptic Ulcer is defined by oxford concise medical dictionary as a breach in the

protective lining (mucosa) of the digestive tract caused by digestion of abnormally

high concentration of pepsin and acid or other mechanism reduces normal

protective mechanism of the mucosa. It is a break in the wall or lining of the

stomach, the duodenum or occasionally the lower esophagus resulting from

digestive action of the gastric juice on the mucus membrane when the latter is

susceptible to its action (Elizabeth, 2010). The most common symptoms are

abdominal pain at meal time referred to as Stomach or Gastric ulcer and duodenal

ulcer, pain at three hours after meal. Other symptoms include; bloating, abdominal

fullness, nausea, copious vomiting and loss of appetite, weight loss, foul smelling

faeces (melena) and acute peritonitis (Bnat and Griram, 2013). Complications may

include bleeding, vomiting of blood (hematemesis), perforations and blockage of

the stomach (Milosavljevic et al., 2011).

Nations of the world are blessed with flowering plants. Plants have been selected

and used as drugs over centuries (salkat et al., 2009). Plants have great potential to

treat both human and livestock diseases (Alawa et al., 2003). 70% of the

population in rural India is dependent on the traditional system of medicine. The

use of herbal medicine in Western world is steadily increasing. 80% of the

population in Africa uses traditional medicine for primary health care (IUCN

Species Commission, 2007). Some herbal medicines have been demonstrated to be

efficacious (MacDonald et al., 2004; Patel, 2012).

Although, herbal medicines are natural, they must be subjected to necessary

investigation to dissect valuable herbal drugs from harmful and toxic ones

(Phlomena, 2011; Efferth et al., 2011). Over the past two decades, there has been a

tremendous increase in the use of herbal medicine, (WHO). The pharmacological

treatment of disease began long ago with use of herbs (Schulz et al., 2001). In

many developing countries, a large proportion of the population relies on

traditional practitioners and their armamentarium of medicinal plants in order to

meet health care needs (Shaw, 1998). Traditional Chinese medicine has been used

by them from time immemorial. Out of more than 12,000 items (animals, mineral

salts and plants) used, the primary source of remedies is botanical (Li, 2000).

Herbs native to Japan were classified in the first Pharmacopoeia of Japanese

traditional medicine in the ninth century (Morgan, 2002). Introduction of

traditional herbal medicines into Europe, the USA and other developed countries

started during the latter part of the twentieth century. The desire to capture the

myth of traditional healing system has led to a resurgence of interest in herbal

medicine (Tyler, 2000). Ayurveda is the system of medicine practiced in India for

the past fifty decades (Morgan, 2002). In Canada, herbal use has also increased

(Berger, 2001). Human beings have used plants for the treatment of diverse

ailments for thousands of years (Sofowora, 1982; Hill 1989). Herbal medicines are

relatively safer and cheaper than synthetic or modern medicine (Iwu et al.,

1999;Idu et al., 2007; Mann et al., 2008; Amara et al., 2009). Researchers have

identified number of compounds used in mainstream medicine which were derived

from ethno-medical plant

Liver damage is a metabolic disorder which is the most common cause of

mortality and morbidity worldwide. Liver injury treatments have gained global

attention in recent times due to many allopathic medications and their toxic effects

which cause liver damage. Hence, folkloric herbs with hepatoprotective potentials

to treat liver injury or disease have attracted considerable attention from

researchers majorly because of their low toxicity and healing efficacy. In recent

times, several folkloric herbs have been assessed for their hepatoprotective role in

experiments on animals. Phytotherapy have been utilized for the benefit of the

beneficial results of herbal medicine for human health. Varieties of molecules have

been analysed and their physicochemical and pharmacological properties were

elucidated. However, compounds and extracts need to be appropriately formulated

to enhance their physiological goal and pharmacological potential. Low

permeability and solubility are factors that could affect the absorption and delivery

of bioactive molecules (Fang and Bhandari, 2011). On the other hand, the shelf life

of herbal drugs should be elucidated to monitor their stability during the period of

use. Degradation reactions are facilitated by temperature, humidity, pH, oxygen,

and light. Herbal medicines are complex mixtures of different classes of

biochemical compounds, such as carbohydrates, lipids, proteins, and secondary

metabolites (Bott et al., 2010). Due to the negative effect of synthetic drugs, much

effort has been made to discover novel sources of hepatoprotective agents (Mamat

et al., 2013). In recent times, most of the hepatoprotective drugs available in the

market for use against different kinds of liver diseases have phytochemical in

origin, either as single-plant preparations or as poly-herbal mixtures. Folkloric

herbs play an important role in improving the quality of life of rural inhabitants,

especially in developing countries with poor or few modern health facilities

(Abhilash et al., 2014). Over 70,000 plant species have been used for therapeutic

purposes. Although attention has been diverted to plants as resorts for natural

treatment due to efficacy, the scientific rationale behind the plant preparation and

dosage regimens has usually not well understood, and despite their efficacy and

cost-effectiveness, the need to ensure low-toxicity candidate plants is of utmost

importance, and although these herbal medicine are abundant in the market and the

pharmacological components have not been fully identified, some of the identified

bioactive components have been confirmed to have antiviral, antioxidant,

anticarcinogenic, antifibrotic, and anti-inflammatory effects

(Olakunle,2011;oloyede et al.,2013)

Ageratum conyzoides is called goat weeds in English, Imi-esu among yorubas,

Alkaura-tuturuwa in hausa and Obiarakara among Igbo speaking people of Nigeria.

It belongs to the family of Asteraceace. It is a pan-tropical herb with a long history

of traditional medicinal uses. Its toxicity has not been well studied but the

extracted oil has a powerful nauseating odour (Sood, 1973). It is an erect aural

branched, slender, hairy and aromatic herb which grows to approximately 1m in

height. It is used as fish feed (Menut et al., 1993). Ageratum conyzoides is an erect

annual herbaceous plant, 30-80cm tall with a long history of traditional medicinal

uses in several countries of the world.

Vernonia amygdalina, a member of the Asteraceae family, is a small shrub that

grows in tropical Africa. V. amygdalina typically grows to a height of 2–5 m (6.6–

16.4 ft). The leaves are elliptical and up to 20 cm (7.9 in) long. The leaves are

green with a characteristic odour and a bitter taste. No seeds are produced and the

tree has therefore to be distributed through cutting. Its bark is rough and it is

commonly called bitter leaf in English because of its bitter taste, Ewuro in Yoruba,

Shakwa shuwaka in Hausa and Onugbu among Igbo speaking people of Nigeria.

Other African common names include grawa (Amharic), Etidot (Ibibio), Ityuna

(Tiv), Oriwo (Edo), Mululuza (Uganda), Labwori (Acholi), Olusia (Luo) and

Ndoleh (Cameroon). In Nigeria, V. amygdalina is used for food and medicinal

purposes. The roots and twigs are used for abdominal and other gastrointestinal

problems in humans while the decoctions from the leaves are used as anti-malaria

in Guinea and as cough remedy in Ghana (Watt and Breyer-Brandwijk, 1962;

Akinpelu, 1999; Masaba, 2000).widely described by livestock farmers as a potent

anti-helmitic (Alawa et al., 2008). The cooked leaves are a staple vegetable in

soups and stews of various cultures throughout equatorial Africa. Traditionally, the

roots of Vernonia amygdalina are used in cleaning teeth by chewing them into

brush-like ends. Investigations have shown those chewing sticks contains some

active substances which are antimicrobial in nature (Sofowora, 1993). Sofowora,

1982 also revealed the antifungal properties of petroleum extract of Heteromorpha

infoliata leaves against cladosporium and ethanolic extracts of Mormodica

chanrantia, Alstonia booner and Ocimum bacilium possessing antimicrobial

activity against Esherichia coli, Salmonella paratyphi and Shigella dysenteriae.

Caution must be displayed in harvesting Vernonia amygdalina for food and

medicinal use. The researchers discovered the oxytoxic activity of Vernonia

amygdlina in hastening parturition.

CITRUS AURANTIFOLIA (LIME TREE)

It belongs to the family Rutaceae. It is used for nausea, indigestion and

constipation (Bent and Nko, 2004). It exhibits activities for cold fevers, sore

throats, sinusitis and bronchitis as well as helping asthma (Khanc, 2007). The Key

lime (Citrus aurantiifolia) is a citrus hybrid (C. micrantha x C. medica) with a

globose (spherical shaped) fruit, 2.5–5 cm in diameter (1–2 in), that is yellow when

ripe but usually picked green commercially. It is smaller and seedier, with a higher

acidity, a stronger aroma, and a thinner rind, than that of the Persian lime (Citrus

latifolia). It is valued for its unique flavor compared to other limes. The name

comes from its association with the Florida Keys, where it is best known as the

flavoring ingredient in Key lime pie. It is also known as West Indian lime,

bartender’s lime, Omani lime, or Mexican lime, the last classified as a distinct race

with a thicker skin and darker green color. Philippine varieties have various names,

including dayap and bilolo.

The English word "lime" was derived, via Spanish then French, from the

Arabic word ليمة līma (Persian: لیمو limu). "Key" is from Florida Keys, where the

fruit is naturalized. The Oxford English Dictionary dates the first use of "key lime"

to 1905, in an issue of Country Gentleman, which described the fruit as "the finest

on the market. It is aromatic, juicy, and highly superior to the lemon." C.

aurantiifolia is a shrubby tree, to 5 m (16 ft), with many thorns. Dwarf varieties

exist that can be grown indoors during winter months and in colder climates. Its

trunk, which rarely grows straight, has many branches, and they often originate

quite far down on the trunk. The leaves are ovate, 2.5–9 cm (0.98–3.54 in) long,

resembling orange leaves (the scientific name aurantiifolia refers to this

resemblance to the leaves of the orange, (Citrus aurantium). The flowers are

2.5 cm (0.98 in) in diameter, are yellowish white with a light purple tinge on the

margins. Flowers and fruit appear throughout the year, but are most abundant from

May to September in the Northern Hemisphere. When in contact with the skin, the

Key lime can sometimes cause phytophotodermatitis, in which a chemical reaction

makes the skin extra sensitive to ultraviolet light.

This particular cultivar is a citrus hybrid, likely Citrus micrantha x Citrus

medica (a papeda-citron cross) (Nicolosi et al., 2000). C. aurantiifolia is native to

Southeast Asia. Its apparent path of introduction was through the Middle East to

North Africa, then to Sicily and Andalucia and via Spanish explorers to the West

Indies, including the Florida Keys. From the Caribbean, lime cultivation spread to

tropical and subtropical North America, including Mexico, Florida, and later

California. Since the North American Free Trade Agreement came into effect,

many Key limes on the US market are grown in Mexico, Central America and

South America. They are also grown in Texas, Florida, and Californi

Citrus aurantifolia has been subjected to extensive phytochemicals,

pharmacological and clinical investigation in the area of insecticides (Loius),

cardiac diseases, anti-cancer, eye conditions, inflammatory bowel disease and

improved lung function (Katrine Baghurst,2003).

The liver is a reddish brown organ with four lobes of unequal size and shape. It

is a vital organ present in vertebrates and some other animals regarded as triangular

organ. It is the largest and most complex internal organ and gland in the human

body (Sembulingam and Sembulingam, 2006). The human liver normally weighs

1.44-1.66kg (Contran et al., 2005; Sembulingam and Sembulingam, 2006). It is a

soft organ located in the right upper quadrant of the abdominal cavity, resting

below the diaphragm, overlies the gall bladder and lies to the right of the stomach.

The liver is necessary for survival; there is currently no way to compensate for the

absence of liver function long term, although liver dialysis can be used short term

(Liver diseases are regarded as one of the leading global health issues and is

majorly prevalent in the developing countries of the world. These diseases are

classified into different categories, namely hepatosis (non-inflammatory), acute or

chronic hepatitis (inflammatory), and cirrhosis or fibrosis (degenerative). The

heavy metals, toxins, malnutrition, and drug abuse usually cause them.

Consequently, the factors mentioned above destroy and incapacitate the

hepatocytes that ultimately result in hepatitis, jaundice, liver fibrosis, and alcoholic

liver disease. The elevation of cholesterol in the bloodstream is one of the

indicators of the liver injury/disease. High percentage of low-density lipoprotein

cholesterol (LDL-C) and triacylglycerols (TAGs) are predisposing factors of

cardiovascular diseases (Dominiczak, 2005).

In addition, the damage of the hepatocytes can also be caused by excessive

consumption of alcohol, toxic substances such as thioacetamide (TAA), abuse of

certain drugs such as paracetamol (PCM), chemotherapeutic agents such as carbon

tetrachloride (CC14), and also some organic and inorganic compounds, aflatoxin,

microbes, and viral infections (for example, hepatitis A, B, C, and D), which have

been adequately investigated (Sathesh et al.,2009; Partap et al.,2014; Ajboye

etal.,2010). The endoplasmic reticulum and mitochondrial cytochrome P-450

metabolize CC14 that subsequently generates reactive oxygen species (ROS,

CCl3O−), which results in a chain reaction and perhaps triggers lipid peroxidation.

Paracetamol (PCM) is commonly used as a pain-relieving (analgesic) or fever-

preventing (antipyretic) drug. The abuse of this drug damages the hepatocytes and

consequently leads to liver cell injury or disease (Jannu et al.,2012). In addition,

the excessive administration of PCM can lead to the death of most of the liver cells

(necrosis) indicated by nuclear pyknosis and eosinophilic cytoplasm, which

consequently results in large excessive hepatic injury. When PCM is metabolized

in the liver, it generates an oxidative product, N-acetyl-P-benzoquinoneimine that

forms a covalent bond with the sulfhydryl groups of protein (cytochrome P-450

enzymes). This process consequently leads to glutathione (GSH) lipid peroxidative

degradation which results in then necrosis of hepatocytes. TAA is another

chemical substance that obstructs the free flow of RNA between the nucleus and

cytoplasm that consequently leads to membrane damage. Thioacetamide (TAA)

metabolite is accountable for this hepatic injury TAA has the ability to deplete

hepatocytes as well as deplete the frequency of oxygen consumption. In addition, it

reduces the volume of bile as well as its contents that are bile salts and deoxycholic

acid. Hepatotoxin associated liver injury makes excretion of bile defective that is

indicated by an increase of serum levels in toxins (Zeashan et al., 2008). Thus,

maintaining the integrity of the liver at all times is indispensable for good human

health (Pradhan and Girish., 2006; Haidry and Malik.,2014; Guo et al.,2017).

Regardless of its extensive regenerative potential, incessant exposure to

environmental pollutants such as chemotherapeutic agents and xenobiotics could

deplete and impair the normal protective ability of the liver, thereby resulting in

liver dysfunction and consequently liver damage (Ali and Kumar., 2015).

On the other hand, the majority of the hepatotoxic agents damage hepatocytes and

subsequently impair the kidney function mostly through lipid peroxidation or other

oxidative forms. In cases of liver damage, the amount of the natural antioxidant

system is inadequate. ROS are generated by environmental substances such as X-

rays, pollutants, ultraviolet radiation, or metabolic process in the mitochondria

(Haque et at., 2014).The intracellular concentration of ROS solely depends on the

rate at which they are generated by exogenous or endogenous factors as well as

their elimination by several endogenous antioxidants such as enzymatic and non-

enzymatic processes (Haque et al., 2014).

Several reports have shown that oxidative stress triggered by free radicals is the

main causative agent of liver damage such as degeneration, necrosis, swelling, and

apoptosis of the hepatocytes. Liver injury or damage caused by free radicals

usually occurs through the mechanisms of lipid peroxidation and covalent bonding

with consequent tissue injury. ROS which include peroxyl, hydroxyl, alkoxy, and

superoxide radicals destroy the membrane lipids, proteins, and nucleic acid, and

this has also been implicated in several aging-related issues together with

atherosclerosis, diabetes mellitus, lung and kidney damage, liver disorders, cancer,

inflammatory diseases, and cardiovascular diseases (Singh et al.,2008; Pal et

al.,2014). Lipid peroxidation affects cell membranes and consequently affects the

structural integrity and functionality of the cell membrane that subsequently

impairs the cell's potential to maintain constant ion gradients and transport

(Madkour and Abdel-Daim., 2013). On the other hand, liver damage can also be

caused by drug abuse in overdosage and certain chemicals (Ali and Kumar, 2015).

Cystic lesions of the liver are commonly encountered in abdominal imaging.

Attention to imaging characteristics and correlation with clinical history and

laboratory findings will permit an accurate diagnosis and appropriate patient

management (Del Poggio et al., 2008). Cystic liver lesions one of the most

common liver lesion, also called fluid-containing lesions of the liver, are

commonly encountered findings on radiologic examinations that may represent a

broad spectrum of entities ranging from benign developmental cysts to malignant

neoplasms. The wide range of pathologic processes that may result in cystic liver

lesions can present a difficult diagnostic conundrum (Murphy et al., 1989). The

radiologist must carefully assess such imaging features as location, size, and

unifocal or multifocal nature of the cyst or cysts as well as evaluate cyst

complexity and associated findings. In addition, because radiologic features of

various cystic liver lesions overlap, it is necessary to integrate imaging with

clinical and laboratory findings to allow more definitive diagnosis (Anderson et al.,

2009). An important first step in narrowing the differential diagnosis is to

determine the presence or absence of complex features in cystic liver lesions. To

this end, fluid-containing liver lesions can be grouped broadly into simple or

complex cysts (Singh et al., 1997).

Simple cysts appear as fluid-containing lesions with smooth thin walls and no

evidence of complex internal features, such as septation and mural irregularity or

nodularity. Simple cysts may be solitary or multifocal. The differential diagnoses

for simple hepatic cysts include benign developmental hepatic cyst, biliary

hamartoma (von Meyenburg complex), Caroli disease, and autosomal polycystic

liver disease (Del Poggio et al., 2008).

Complex cysts are fluid-containing hepatic lesions with one or more of the

following complex features: wall thickening or irregularity, septation, internal

nodularity, enhancement, calcification, and hemorrhagic or proteinaceous contents.

Because a broad range of disease processes can result in complex cystic liver

9lesions, they may be further grouped as neoplastic, inflammatory or infectious,

and other miscellaneous entities (Ghai et al., 2005). A careful evaluation of

particular imaging features as well as associated radiologic and clinical and

laboratory findings is necessary to suggest a specific diagnosis (Mortele et al.,

2001). Currently, excessive alcohol consumption is one of the major causes of liver

disorders globally (Ali and Kumar, 2015)

There is a nexus between ethanol intake and alcoholic liver disease because

alcohol metabolism takes place in the liver, and consequently, this process affects

lipoprotein and lipid breakdown.

Furthermore, alcohol dehydrogenase converts ethanol to acetate that generates

ROS through cytochrome P4502E1 (Wahid et al.,2016; Lu and Cederbaum.,2008)

The process that occurs in the liver results in oxidative stress and subsequently

brings about hepatic damage and affects the structural rigidity of the hepatic cell

membrane which allows the cytosolic enzymes to escape into the bloodstream.

Consequently, the biochemical marker commonly used to detect liver damage is an

increase in the cytosolic enzymes in the bloodstream (Ramaiah, 2007).

Concentrations of aspartate transaminase (AST) and alanine transaminase (ALT) in

the cytoplasm and mitochondria of damaged hepatocytes also increase. Leakage of

the cell membrane causes an increase in serum hepatospecific enzymes due to the

distortion of the liver cell membrane structure. In addition, hyperbilirubinaemia is

a manifestation of an increase rate of erythrocyte breakdown. Thus, the

conservation of a healthy liver is indispensable for good human health (Fang and

Bhandari, 2011).

Working Hypothesis

Ho- The two anti-ulcer herbal mixtures, Idogun and Adedironic DO NOT possess

hepato-protective potentials at both low and higher concentrations

H1- The two anti-ulcer herbal mixtures, Idogun and Adedironic possess hepato

protective potentials at both low And higher Concentrations.

MATERIALS AND METHODS

Idigun anti-ulcer mixture prepared by the herbal practitioner has the following

content. Ageratum conyzoides plants, Vernonia amyzdalinch roots, Citrus

aurantifiola root, palm oil, palm kernel oil, Shea butter and Otubuyo. The second

herbal mixture was prepared by the researchers and it contains Ageratum

conyzoides plants, vernonia amygdalina roots and roots of Citrus auran tifiola.

These were thoroughly washed and dried at room temperature for four weeks.

They were separately crushed and reduced to fine powder using domestic electric

grinder. They were mixed together; 854g Ageratum conyzoides, 183g of Vernonia

amygdalina and 183g of Citrus aurantifiola and dissolved in 15litres of 70%

alcohol. This was left at room temperature for 3days, filtered using whatman filter

paper. The filtrate was concentrated at 400/c in waterbath for two weeks. The

extract was packed in brown bottles and refrigerated at 40/c. 2000g of idogun

mixture was equally dissolved in 15litres of 70% alcohol and treated like the

former. The two herbal mixtures were administered separately at two settings, the

low and higher concentrations.

Preparation of Test Animals

50 rats of either sex were bread locally and acclimatized for two weeks. They were

housed in standard plastic cages, fed with standard pellet diet and allowed free

acess to water. All animals received humane care in accordance with international

guildelines (national research council of the national acedemics 2011)

The former has 6 groups of 5 rats; the control, 100mg/kg, 200mg/kg, 400mg/kg,

800mg/kg and 20mg/kg omeprazole. The second set is made of 4 groups with 5

rats per group. They are the control, 500mg/kg, 1000mg/kg and 2500mg/kg of both

herbal mixtures. The experimentation for low concentrations took 14days while

higher concentrations, 28days.

The rats in lower concentration groups were sacrificed 1 hour after oral

administration of 1ml absolute alcohol to induce injury in the gastric mucosa, the

liver organs were harvested and fixed in 10% neutral buffered formalin. The blood

sample of the rat in higher concentration groups were collected from their

abdominal aortas after anesthesia for chemical analysis. The lower organs were

also harvested and treated like the former. The liver organs were processed and

stained with heamatoxylin and eosin stains before microscopical analysis. The liver

function test ran on the blood sample direct bilirubin, total bilirubin, total protein,

albumin, aspartate amino-transfarace (ASP) and alanine amino-transferase (ALT).

Prepared Idogun anti-ulcer herbal mixtures with written method of its

preparations were submitted by the herbal practitioner while the adedironic was

prepared by the researchers.

Preparation of Adedironic Anti-Ulcer Herbal Mixture

Whole plants of Ageratum conyzoides, roots of Vernonia amygdalina and Citrus

aurantifolia used were identified and registered with the department of botany,

Faculty of Life Sciences, University of Benin, Benin City with registration

numbers UBHA286, UBHC288 and UBHV287.

They were washed in running water to remove all dirts and air-dried for

4weeks (Nora et al., 2016). They were separately crushed in a clean mortar with

pestle and later reduced to fine powder by grinding using domestic electric blender.

The granules were measured as specified by the herbal practitioner, 35g of

Ageratum conyzoides, 7.5g of Vernonia amygdalina and 7.5g of Citrus

aurantifolia. They were thoroughly mixed together.

RESULT

SUMMARY OF HEPATO-PROTECTIVE PROPERTY OF BOTH IDOGUN

(H) AND ADEDIRONIC (P) HERBAL MIXTURES.

Dose Liver

Control Balooning degeneration

H 100mg/kg Normal Histology

P 100mg/kg Normal Histology

H 200mg/kg Parenchymal haemorrhage

P 200mg/kg Normal Histology

H 400mg/kg Normal Histology

P 400mg/kg Normal Histology

H 800mg/kg Normal Histology

P 800mg/kg Normal Histology

H Omeprazole Fresh Parenchyma haemorrhage

PLATES 19-24: LIVER (19) A, Balooning degeneration (20) Normal (21) Parenchymal

haemorrhage (22) Normal (23) Normal (24) A, Fresh parenchymal

2019

A

2221

A

2423

A

hemorrhage 19-24 (Control, 100, 200, 400, 800mg/kg Idogun concoction and

20mg/kg Omeprazole groups respectively). H&E x400

39 40

4241

A

3837

A

PLATES 37-42: LIVER (37) A, Fresh haemorrhage with the liver Parenchymal (38)

Normal (39) Normal (40) Normal (41) Normal (42) A, Fresh haemorrhage

into the parenchymal 37-42 (Control, 100, 200, 400, 800mg/kg Plant mixture

and 20mg/kg Omeprazole groups respectively). H&E x400

COMPARISON OF THE EFFECT OF HIGHER DOSES OF BOTH

ADEDIRONIC (P) AND IDOGUN HERBAL MIXTURES (H) ON THE

LIVER.

DOSES LIVER

CONTROL Vascular Congestion

H 500mg/kg Normal Histology

P 500mg/kg Normal Histology

H 1000mg/kg Normal Histology

P 1000mg/kg Baloon degeneration of hepatocyte

H 2500mg/kg Balooning degeneration of hepatocyte

P 2500mg/kg Fatty Change

PLATES 65-68: LIVER (65) Normal (66) Normal (67) A, Balooning degeneration of

hepatocytes (D) A, Vascular congestion 65-68 (500, 1000, 2500mg/kg Idogun

concoction and CONTROL groups respectively). H&E x400

65 66

67 68A

A

PLATES 69-72: LIVER (69) Normal MASSON TRICHROME X400 (70) Normal PAS

X400 (71) A, Balooning degeneration of hepatocytes PAS X400 (72) A,

Vascular congestion PAS X400 69-72 (500, 1000, 2500mg/kg Idogun

concoction and CONTROL groups respectively).

69 70

71 72

A

A

AST ALT0

10

20

30

40

50

60

47

22.6

48

14

49.6

24.4

42.4

21.2

CONTROL(0.5g/kg)(1g/kg)(2.5g/kg)

CON

CEN

TRAT

ION

(IU

/L)

FIG: AST AND ALT IN TEST AND CONTROL RATS – HERBAL

MIXTURE

TABLE 1

LIVER FUNCTION TESTS FOR IDOGUN HERBAL MIXTURE

Direct

bilirubin

(DB)

Total

bilirubin (TB)

Total

protein(TP)

Albumin(Alb)

CONTROL 0.1±0.04 0.6±0.2 6.4±0.6 3.5±0.4

500mg/kg 0.4±0.2 0.8±0.2 7.0±0.3 3.7±0.2

1000mg/kg 0.3±2* 0.7±0.3 6.5±0.4 3.4±0.1

2500mg/kg 0.2±0.1 0.6±0.4 6.8±0.6 3.7±0.3

Statistically significant at (P˂0.05)

Key- DB- Direct bilirubin concentration TB-TB concentration, TP-Total protein

concentration, ALb-Albumin concentration.

Note- Results are presented as mean ± standard deviation.

TABLE 2

LIVER FUNCTION TESTS FOR ADEDIRONIC HERBAL MIXTURE

Direct

bilirubin

(DB)

Total

bilirubin (TB)

Total

protein(TP)

Albumin(Alb)

CONTROL 0.1±0.1 0.8±0.2 6.5±0.8 3.4±1.4

500mg/kg 0.3±0.2 0.6±0.4 6.5±0.7 3.5±0.5

1000mg/kg 0.4±0.2 0.8±0.3 5.6±2.4 3.4±0.2

2500mg/kg 0.3±0.2 0.7±0.4 6.8±0.3 39±0.1

Key- DB- Direct bilirubin, TB total bilirubin, Total protein and ALb-Albumin

Note- Results are presented as mean ± standard deviation.

AST ALT0

10

20

30

40

50

60

70

52.4

24.2

47.8

18

59.4

36.2

49

18.8

CONTROL(0.5g/kg)(1g/kg)(2.5g/kg)

CON

CEN

TRAT

ION

(IU

/L)

FIG: AST AND ALT IN TEST AND CONTROL RATS – PURE

PLANT MIXTURE

DISCUSSION.

The two herbal mixtures were found to possess hepatocellular protective

potentials as no inflammation or hepatitis was noted after administration of

both low and higher concentrations unlike Germander furano diterpenoids that

caused apoptosis, DNA fragmentation and over expression of p53.( Fau etal),

1997. Both herbal mixtures (Idogun and Adedironic) at high concentrations do

not impair bilirubin clearance. This is in tandem with Wendong et al;2004

where a decoction of Yin chin and three other herbs proved to be a key

regulator of bilirubin clearance in liver. The administration of Idogun and

Adedironic herbal mixtures promote protein synthesis as seen in table 2, this

will help in regeneration of liver tissue, prevention of hepatitis and

maintenance of glutathione level. This is in agreement with Pradhan and

Girish, 2007 where silymarin,, a flavonolignan from Silybum marianum is

used and adapted for hepato-protection because of its antioxidative, anti-lipid

peroxidative and anti-inflamatotory properties. At 1g/kg of Adedironic

mixture, an increased ALT and AST was observed just like in Graham et al,

1994 which showed hepatotoxicity associated with ingestion of the Chinese

herbal product Jin Bu Huan tablets.

The histomorphology of the liver for the two herbal mixtures at low

concentrations showed protection of the integrity of the liver cells\hepatocytes,

this is supported by sakanal et al 2003 in which administration of indian-ko-to

[Tj-135] prevents liver fibrosis and enzyme-altered lesions.

CONCLUSION

The study has revealed that both Idogun and Adedironic herbal mixtures

at low and higher concentrations possess hepatoprotective properties. Further

studies are required to unravel the effect of the two extracts on hormonal

profile of Albino rats of either sex.

REFERENCES

Ajiboye TO, Yakubu MT, Salau AK, Oladiji AT, Akanji MA, Okogun JI, et al. Antioxidant and drug detoxification potential of aqueous extract of Annona senegalensis leaves in carbon tetrachloride-induced hepatocellular damage. Pharm Biol 2010;48:1361-70.

Abhilash G, Maheswari YJ, Gopal A, Chanda D. Review on some medicinal plants with hepatoprotective activities. Res Rev J Pharmacogn Phytochem 2014;22:33.

Ali MI, Kumar M. A recent update on hepatoprotective potential of herbal plant. Int J Env Sci

Alawa CBI, Adamu AM Gefu JO, Ajanusi DJ Jagin AJ, Lamidi OS and Oni OO (2008): Ethnoveterinary practices in Nigeria survey of plants used as anthelmintics.VomJournalVetenary Sciences5:1-11.

Amara A, El-Masry MBogdady H (2009): Plant Crude Extracts could be the Solution; Extract showing in vivo Anti-tumorigenic activityJournal of Pharmacological Science.21(2):159-171.

Anderson SW, Kruskal JB, Kan RA (2009): Benign hepatic tumors and iatrogenic pseudotumors. RadioGraphics 2009; 29:211–229.

Akinpelu DA (1999): Antimicrobial activity of vernonia amygdalina leaves. Fitoterapia,(70):432-434.

Bent S and Nko R (2004): Commonly used herbal medicines in the United States- a review. America Journal of Medical Genetics116:478-485.

Bnat and Griram (2013). SRB’s Manual for Surgery. (364).

Bassam Abdul Rasool Hassan (2012): Medicinal Plants (Importance and Uses). Pharmaceutica Analytical Acta Journal (3):139.

Bott RF, Labuza TP, Oliveira WP. Stability testing of spray- and spouted bed-dried extracts of Passiflora alata. Dry Technol 2010;28:1255-65.

Berger Population Health Monitor Survey (2001): Government of Canada.

Del Poggio P, Buonocore M, (2008): Cystic tumors of the liver: a practical approach World J Gastroenterol; 14:3616–3620.

Dominiczak MH. Lipids and Lipo proteins. In: Baynes JW, Dominiczak MH, editors. Medical Biochemistry. Philadephia: Elsevier Mosby; 2005. p. 234-42.

Elizabeth AM (2010): Peptic ulcer, Oxford Concise Medical Dictionary, eight edition. Oxford University press Inc. new york ISBN; 978-0-19-955714-1

Efferth T and Kaina B (2011): Toxicities by herbal medicines with emphasis to traditional Chinese medicine. Current DrugMetabolism (10):989-996.

Eloff JN (1995). Which Extract should be used for the Screening and Isolation of Antimicrobial Components from plants.Journal of Ethnopharmacology60 (1):1-8.

FabricantDSand Farnsworth NR (2001): The value of Plants used in Traditional Medicine for Drug Discovery. Environmental Health Perspectives (109):69-75.

Fang Z, Bhandari B. Effect of spray drying and storage on the stability of bayberry polyphenols. Food Chem 2011;129:1139-47.

Fau D, Lekehal M, Farrell, G, Morau A, Mouly C, Feldmann G, Hauuzi D and Passayre D (1997). Diterpenoid from Germander an herbal medicine, induce apoptosis in isolated rat hepatocytes gast. 113 (4): 1334-1346

Ghai S, Dill-Macky M, Wilson S, Haider M. (2005): Fluid- fluid levels in cavernous hemangiomas of the liver: baffled? American Journal of Roentgenology; 184:82-85.

Graham MW, Lidia MP, Sergio ER, Sharrilyn W, Federico GV, William NK, Pina M Ian RW Frank RS, John JB and John MV (1994) Acute Hepatitis Assiciated with the chinese herbal product Jun Bu Huan Ann Intern med.121 (10): 729-735

Guo S, Guo T, Cheng N, Liu Q, Zhang Y, Bai L, et al. Hepatoprotective standardized EtOH-water extract from the seeds of Fraxinus rhynchophylla hance. J Tradit Complement Med 2017;7:158-

Haque A, Tahmina, Afsana SK, Sarker IR, Hossain M, Islam S, et al. Antioxidant and hepatoprotective effects of aqueous

and ethanol extracts of Dendrophthoe falcate Linn leaves. Archives 2014;1:90-101

Haidry M, Malik AT. Hepatoprotective and antioxidative effects of Terminalia arjuna against cadmium provoked toxicity in albino rats (Ratus norvigicus). Biochem Pharmacol 2014;3:1.

Hill AF (1989). Economic Botany; a Text book of Useful Plants and Plant Products, 2nd ed. Ml Graw Hill Book Company, Inc, New York. Pg560.

Iwu MM, Duncar AR, Okungi CO (1999): New Antimicrobials of Plant Origin. In Janick, J.(ed) Perspectives in New Crops and New Uses. ASHS Press, Alexandria, V.A. pp 457-462.

Idu M, Omogbai EKI, Aghimien GEI, Amaechina F, Timothy O and Omonigho SE (2007): Preliminary Phytochemistry Antimicrobial properties and Acute Toxicity of Stachytarphapheta jamaicensis (L) Vahl leaves. Trends in Medical Research (2):193-198.

Jannu V, Baddam PG, Boorgula AK, Jambula SR. A review on hepatoprotective plants. Int J Drug Dev Res 2012;4:1-8.

KuipersEJ, Thijs JC, Festen HP (1995): The Prevalence of Helicobacter pylori in Peptic ulcer Disease. Alimentary Pharmacology Therapeutics(2):59-69.

Khanc CP (2007): Indian medicinal plants. An illustrated dictionary.Spring publication.Pg153-157.

Lu Y, Cederbaum AI. CYP2E1 and oxidative liver injury by alcohol. Free Radic Biol Med 2008;44:723-38.

Li L (2000): Opportunity and Challenge of Traditional Chinese Medicine in face of the Entrance to WTO (World Trade Organization). Chinese Information Traditional Chinese Medicine (7):7-8.

Langman MJS, Wal J, Wain wright P, Lawson DH, Rawlins MD, Logan RFA, Murphy M, Vessey MP and Collin Jones DJ (1994). Risks of bleeding Peptic ulcer associated with individual non-steroidal Anti-inflammatory Drugs. The Lancet (343):1075-1078.

Milosavljevic T, Kostic-Milosavljeric M, Jovanovic I and Krotic M (2011): Complications of peptic ulcer disease. Digestive Diseases 29(5):491-493.

Masaba C(2000). The antimalarial activity of Veronia amygdalina Del(composite).Transaction of the Royal Society of Tropical Medicine and Hygiene Journal.(94):694-695.

MacDonald K, Vancrey K and Harrisson P (2004): In vitro genotoxic evaluation of the medicinal plant. Journal of ethno-pharmacology. (81):11-16.

Mamat SS, Kamarolzaman MF, Yahya F, Mahmood ND, Shahril MS, Jakius KF, et al. Methanol extract of melastoma malabathricum leaves exerted antioxidant and liver protective activity in rats. BMC Complement Altern Med 2013;13:326.

Madkour FF, Abdel-Daim MM. Hepatoprotective and antioxidant activity of Dunaliella salina in paracetamol-induced acute toxicity in rats. Indian J Pharm Sci 2013;75:642-8.

Mann A, Banso A, Clifford LC (2008): Antifungal properties of Anogeissus leiocarpus and Terminalia aricennioides. Tanzania Journal of Health Research (10):34-38.

Murphy BJ, Casillas J, Ros PR, Morillo G, Albores- Saavedra J, Rolfes DB. (1989): The CT appearance of cystic masses of the liver. RadioGraphics; 9:307–322.

Memut C, Lamaty G and Amvam PH (1993):Flavour Fragrance Journal(8):1

Mortele KJ, and Ros PR (2001): Cystic focal liver lesions in the adult: differential CT and MR imaging features. RadioGraphics; 21:895–910.

Morgan K (2002): Medicine of theBasic principles of Ayurvedic medicine (http://www.compulink.co.uk/-- marodruk/Ayurveda.htm).

Nwachukwu CU, Ume NC, Obasi W, Nzewuche GU and Onyirioha CU (2010): The Quantitative Uses of some Medicinal Plants in Ikeduru L.G.A of Imo State, Nigeria. New York Science JournalVol.3, (11):129-134.

Nicolosi E, Deng ZN, Gentile A, La Malfa S, Continella G and Tribulato (2000): Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theoretical and Applied Genetics 100(8): 1155-1166.

Olakunle Ajiboye T. In vivo antioxidant potentials of Piliostigma thonningii (Schum) leaves:Studies on hepatic marker enzyme, antioxidant system, drug detoxifying enzyme

and lipid peroxidation. Hum Exp Toxicol 2011;30:55-62.

Oloyede HO, Ajiboye TO, Komolafe YO. Polyphenolic extract of Blighia sapida arilli prevents Nnitrosodiethylamine-mediated oxidative onslaught on microsomal protein, lipid and DNA. Food Biosci 2013;1:48-56.

Pal S, Bhattacharjee A, Mukherjee S, Bhattacharya K, Khowala S. Antioxidant and hepatoprotective activity of ethanolic extract of Alocasia indica tuber. Am J Phytomed Clin Ther 2014;2:191-208.

Pradhan SC, Girish C (2007) Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian Journal Med Res (5): 491-504.

Patel DK (2012): Medicinal plants in G.G.V. Campus, Bilaspur, Chhattisgarh in Central India International Journal of Medicine Vol2.(2):293-300.

Pradhan SC, Girish C. Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian J Med Res 2006;124:491-504.

Phlomena G (2011): Concerns regarding the safety and toxicity of medicinal plants. An Overview. Journal of Applied Pharmaceutical Science01(06)40-44.

Partap S, Tewari U, Sharma K, Jha KK. Hepatoprotective activity of whole plant extract of Leptadenia pyrotechnica against paracetamol induced damage in rats. J Drug Deliv 2014;4:36-9.

Ramaiah SK. A toxicologist guide to the diagnostic interpretation of hepatic biochemical parameters. Food Chem Toxicol 2007;45:1551-7.

Rafi Khan Pathan, Rapi Reddi Gali, Parveen Pathan Tanaki Gowtham and Soujanya Pasu Puleti (2012): Invitro Antimicrobial Activity of Citrus aurantifolia and its phytochemical Screening.

Sood, VK (1973): Fkiv ind.4:77.

Sofowora A (1993): Medicinal plants and Traditional Medicines in Africa. Chichester John Wiley and Sons, New York

Sembulingam K and Sembullingan P (2006): Essentials of Medical Physiology. 4th Ed. Jaypee brothers Medical Publishers Ltd.

Singh Y, Winick AB, Tabbara SO (1997): Multiloculated cystic liver lesions: radio-pathologic differential diagnosis. RadioGraphics17:219–224.

Salkat S, Raja C, Biplab D and Joy DM (2009): Plants and phytochemicals for peptic ulcer. An overview.Pharmacognosy review3(6) 270-279.

Singh N, Kamath V, Narasimhamurthy K, Rajini PS. Protective effect of potato peel extract against carbon tetrachloride-induced liver injury in rats. Environ Toxicol Pharmacol 2008;26:241-6.

Schulz V, Hansel R and Tyler VE (2001): Rational Phytotherapy: A Physician’s Guide to Herbal Medicine, 4th Ed., Berlin, Springer-verlag.

Shaw D (1998): Risks or Remedies? Safety Aspects of Herbal Remedies.Journalof the Royal Socirty of Medicine (91):294-296.

Sathesh Kumar S, Ravi Kumar B, Krishna Mohan G. Hepatoprotective effect of Trichosanthes cucumerina var cucumerina L. on carbon tetrachloride induced liver damage in rats. J Ethnopharmacol 2009;123:347-50.

Tyler VE (2000): Herbal Medicine; From the past to the future. Public Health and Nutrition Journal(3):447-452.

Wendong H, Jun Z and David Dm (2004). A traditional herbal medicine enhances bilirubin clearance by activating

the nuclear receptor (AR. Jclin Invest) 113 (1) : 137-143.

Zeashan H, Amresh G, Singh S, Rao CV. Hepatoprotective activity of Amaranthus spinosus in experimental animals. Food Chem Toxicol 2008;46:3417-21.