THE EFFECT OF WONDERFUL COLA ON LIVERHISTOLOGY IN ALLOXAN INDUCED DIABETIC WISTAR

RATS

TABLE OF CONTENTSCover Page - - - - - - - - iTitle Page - - - - - - - - iiCertification - - - - - - - -

iiiDedication - - - - - - - - ivAcknowledgements - - - - - - vTable of Contents- - - - - - - viAbstract - - - - - - - - vii

CHAPTER ONE1.0 Introduction - - - - - - - 11.1 Background of the study - - - - -

11.2 Aim and Objective of the Study - - -

- 31.3 Significance of the study - - - - -

41.4 Definition of Term - - - - - -

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CHAPTER TWO

1

2.0 Literature Review- - - - - - 62.1 Description of Buccholzia coriacea - - - 72.2 Preliminary Studies on Buccholzia coriacea - -

112.3 Liver Physiology - - - - - - 152.4 Liver Anatomy - - - - - - 162.5 Liver Histology - - - - - - 182.6 Functions of the Liver - - - - -

202.7 Disease of the Liver - - - - - 22

CHAPTER THREE3.0 Research Methodology - - - - - 243.1 Experimental Animals - - - - - 243.2 Wonderful Cola Collection and Identification

- 243.3 Temperature and Humidity - - - - 253.4 Preparation of Wonderful Cola Extract- - -

253.5 Induction of Experimental Diabetes- - -

263.6 Experimental Protocols - - - - -

263.7 Blood Collection - - - - - - 273.8 Preparation of Tissue for MicroscopicExamination- 283.9 Statistical Analysis - - - - - -

2

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CHAPTER FOUR4.0 Results - - - - - - - 344.1 Body Weight - - - - - - 34

CHAPTER FIVE5.0 Discussion, Conclusion and Recommendation -

415.1 Discussion - - - - - - - 415.2 Conclusion - - - - - - - 465.3 Recommendation - - - - - - 46References - - - - - - - - 47Appendix I - - - - - - - - 50Appendix II - - - - - - - - 51

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ABSTRACT

Wonderful cola (Buccholzia coriacea) which have been of health

benefit to human, have be screen of diverse pharmacological

activities which of, it has shown an more efficacious effect on diabetic

using animal (wistar) as an experimental tools to checkout its anti-

diabetic effect, on liver histology. This which was carry out using

thirty (30) animal (rat) shared into five (5) group of which group one

was use as control, acclimatize for two weeks and using alloxan to

induce diabetic and the treated with aqueous extract of wonderful

(Buccholzia coriacea). Which glibenclamide were used as standard

drugs to compare with the extract, and dose related. The organ was

harvested (liver) and place on formalin and snap with microscope to

show the affected area of the liver tissue.

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CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study

Plants are primary source of medicine, fibres,

food, shelter, etc. In every day use by humans with

roots, stem, leaves, flowers, and seeds. Providing

food for human diet, supplying the body with

vitamins and certain precursors and calorific

values which makes them necessary in diet

(Codoemelam, 2005). Among these plant seeds are the

seeds of Buccholzia coriacea popularly known as

wonderful kola.

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Buccholzia coriacea is a perennial plant which grows as

a tree. It is an understorey forest tree with large

glossy leathery leaves and conspicuous creamy white

flower. It belongs to the family of capparaceae.

It’s English Name is called musk tree and its local

names include “Uworo” (Yoruba), “Owi” (Edo) are the

seeds which are either cooked or eaten raw

(Quattechi, 2009). It is a brain food which

promotes memory.

According to (Adisa et al., 2010), the seeds are used

traditionally for treating diabetes, hypertension,

rheumatism, cold, cough and catarrh. It prevents

premature aging and has the ability to stop

migraine headache on the fore head for about ten

6

(10) minutes. The stem and barks of the tree

exhibit high concentration dependent antibacterial

and antifungal activity when subject to ethanol

extract (Ajaiyeoba et al., 2009).

Histology: This is the study of the structure of

tissue by means of special staining technique

combined with light and electron microscope. Liver

the largest gland of the body, weighing 1200-1600g,

situated in the top right portion of the abdominal

cavity, the liver is divided by fissures (fossae)

into four lobe; the right (the largest lobe); left

quadrate, and candate lobes, it is connected to

diaphragm and abdominal wall by five ligament.

1.2 Aim and Objective of the Study

7

This study is designed to investigate the

effect of aqueous extract of Buccholzia coriacea on

liver histology in alloxan induced diabetic rat.

This study is also aimed to be of great usefulness

to students who carry out research work on related

topic, so as to provide useful information on

health implication, safe administration and usage

of aqueous extract of Buccholzia coriacea.

A. General Objective: This study aims at checking

the observable effect of aqueous extract of

Buccholzia coriacea.

B. Specific Objective: This study is designed to

investigate the effect of aqueous extract of

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Buccholzia coriacea on liver histology in alloxan

induced diabetes.

This research is conducted in a confined area

under a suitable atmospheric pressure condition.

Thirty (30) rats is used, the rat are fed with

normal rat feed for two week of acclimatization.

Thereafter, the aqueous extract of Buccholzia coriacea

is prepare and administered to the alloxan induce

diabetic rat for four (4) weeks. This research

therefore covers six (6) weeks.

1.3 Significance of the study

This study helps to enumerate possible

beneficial effect of Buccholzia coriacea on liver

histology in alloxan induce diabetic rat.

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1.4 Definition of Term

Liver: The liver is the body largest compound

gland. It is a major metabolic organ, and is

important for degrading so many substance, like

alcohol and drug.

Histology: The study of the structure of tissue by

means of special staining technique combined with

light and electron microscope.

Diabetic Mellitus: Is a chronic metabolic disorder

characterized by a high blood glucose

concentration-hyperglyceamia, caused by insulin

deficiency, often combined with insulin resistance.

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CHAPTER TWO

2.0 LITERATURE REVIEW

The use of herbal medicine (medicinal plant)

has recently gained popularity. Some plants have

basic nutritional importance by their content,

protein, carbohydrate, fat and oil, minerals

vitamin and water which are responsible for growth

and development in man and animal. Some plants

seeds are of crucial medicinal importance in

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treatment of chronic disease such as cancer,

cardiovascular disease and diabetes (Aruoma, 2003).

It has also been discovered that regular

consumption of fruit, vegetable, herbs and spices

has always been associated with health benefit, but

their mechanism of action became clearly only in

recent year. These plant contain a wide variety of

biologically active and non-nutritive compound

known as phytochemical (Sheeta and Jaumuna, 2009).

The extract of Buccholzia coriacea won’t be an

exception.

2.1 Description of Buccholzia coriacea

Buccholzia coriacea is a medicinal perennial plant.

It is an under storey forest tree with large glossy

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leathery leaves and conspicuous creamy white

flowers at the end of the branches (Mojab et al.,

2003). The plant is easily recognized by the

compound pinnate leaves and long narrow angular

fruits containing large usually aligned seeds.

There are about 3-4 seeds in fruits. They are

blackish with a spicy taste. The leaves are large

ellipsoid between 15-25cm long and 5-7.5cm broad

(Keay, 1989).

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Fig 1: Under Storey Forest Tree of Buccholzia coriacea

Fig 2: Leaves of Buccholzia coriacea14

Fig 3: Seed of Buccholzia coriacea Fig 4: Nuts of Buccholzia coriacea

2.1.1 Constituent of Buccholzia coriacea

The phytochemical evaluation of aqueous extract

of air dried seed of Buccholzia coriacea have been shown

to be rich in alkaloids, saponin, glycocides,

steroid, tannin, flavonoid terpernes and reducing

compound (Sugar and phenols).

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2.1.2 Classification of Buccholzia coriacea

Kingdom Plantae

Rhythm: Magnoliophyta

Class: Manoliopsida

Order: Brassicales

Family: Capparaceae

Genus: Buccholzia

Species: Buccholziaacirriaccea

2.1.3 Uses of Buccholzia coriacea

1. It is a brain food which promotes memory

2. It acts as a cleanser of blood.

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3. It is used in the treatment of diabetes,

hypertension, rheumatism, cold, cough (Adisa et al.,

2010).

4. It has analgesic effect (Ezeja et al., 2011).

5. It has antibacterial and antifungi activity

(Ajayeoba et al., 2003; Ezekiel and Onyeozir, 2009).

2.2 Preliminary Studies on Buccholzia coriacea

A lot of work has been carried out using the

leaves, bark, seed and fruit of the plant B.coriacea

(Wonderful cola).

In 2008, the antimicrobial properties of fresh

Buccholzia coriacea (Wonderful cola) and its extract

was investigated. The study showed the

antimicrobial activity of the fresh cola, methanol

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and hexane extract of B.coriacea (Wonderful cola) on

food pathogens. This was evaluated by measuring the

zone of inhibition on nutrient agar for bacteria

and malt extract agar for fungi. The food borne

pathogens used in this study are Escherichia coli,

enterococcus faecalis, staphylococcus aureus, Trichodermaviride and

Aspergillusniger.

The fresh cola was found to be more active on

the test food borne pathogens, the hexane and

methanol extract. The lower inhibitory properties

of the extract confirms that over exposure to air,

sunlight, too much artificial heat and rapid drying

can cause a loss of essential oils. This study

indicates that B.coriacea possesses an invaluable but

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yet to be tapped potentials which, if exploited,

will benefit the food industry (Ezekiel and

Onyeoziri, 2009).

In 2012, an experiment was carried out to study

the effects of methanol extract of Buccholzia coriacea

seed on male reproductive parameters in albino

wistar rats. The study showed that B. coriacea seed

extract significantly increased the testicular

epididymal total tissue protein (P<0.05).

The results suggests that the extract of

Buccholzia coriacea may have anti-fertility effects in

male rats, the site of action most probably the

epididymis (Zhon et al., 2008).

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An experiment was carried out in 2009 to show

the nutritive and anti-nutritive evaluation of

wonderful cola (Buccholzia coriacea) seed. Levels of

some nutrient, anti-nutrient and dietary fibre of

wonderful cola (Buccholzia coriacea) seeds were

determined using standard analytical methods. Crude

protein, crude fat, crude fibre, ash, carbohydrate,

moisture content and calorific values were

obtained. These result reveal that these seeds

contain appreciable amount of nutrient especially

carbohydrates and proteins with good calorific

value and low levels of toxicant and should be

included in human diet to supplement our daily

allowance needed by the body (Ameachi, 2009).

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In 2012, effect of aqueous seed extract of

Buccholzia coriacea (wonderful kola) on the lipid

profile of albino rats was studied. This study

investigated the effect of ethanol extract of

Buccholzia coriacea seeds on the serum lipid profile of

albino rats within a period of two weeks of

treatment.

In conclusion, the results obtained from this

showed that oral administration of aqueous extract

of B.coriacea seeds resulted in a significant

improvement on the levels of serum lipid profile by

a significant increase in HDL concentration,

reducing LDL, Cholesterol concentration without any

practical effect on triacylglycerol and total

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cholesterol. This study suggests that the plant

extract posses hypolipidemic activity, and may be

useful in the management of cardiovascular diseases

and thus suggest that further research on the

extract should be carried out to elucidate the

actual mechanism underlying this effect (Enechi et

al., 2013).

In 2013, the phytochemical quality of dried

seeds of Buccholzia coriacea was studied. The

qualitative and quantitative phytochemical

analyses. Ethanol and methanol extract of dried

seeds of Buccholzia coriacea, were determined by using

standard methods. This study concluded that both

ethanol and methanol extract contained alkaloids,

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glycosides, saponin, tannin, flavonoids, terpernes,

reducing compounds, and phenol qualitatively

(Ibrahim and Fagbohun, 2013).

2.3 Liver Physiology

The liver is a vital organ present in

veterbrate and some other animal. It has a wide

range of functions detoxification, protein

synthesis, and production of biochemicals necessary

for digestion. The liver is necessary for survival,

there is current no way to compensate for the

absence of liver function in the long term,

although new liver dialysis techniques can be used

in the short term. This gland plays a major role in

metabolism and has a number of functions in the

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body including glycogen storage, decomposition of

red blood cell, plasma protein synthesis, hormone

production, and detoxification. It lies below the

diaphragm in the abdominal-pelvic region of the

abdomen. It produces bile and alkaline compound

which aid in digestion via the emulsification of

lipid. The liver highly specialized tissues

regulate a wide variety high-volume biochemical

reaction including the synthesis and breakdown of

small and complex molecules, many of which are

necessary for normal vital function (Maryanna

Quonwarner Wright, 1993).

2.4 Liver Anatomy

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The liver is a reddish brown organ with four

lobes of unequal size and shape. A human liver

normally weighs 1.44-1.66kg and is a soft pinkish-

brown triangular organ. It is both the largest

gland in the human body. It is located in the right

upper quadrant of the abdominal cavity, resting

just below the diaphragm. The liver lies to the

right of the stomach and overlies the gall bladder.

It is connected to two large blood vessel, one

called the hepatic artery and one called the portal

vein. The hepatic artery carries blood from the

aorta, whereas the portal vein carries blood

containing digested nutrient from entire

gastrointestinal tract and also from the spleen and

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pancreas. These blood vessels subdivide into

capillaries, which then lead to a lobule. Each

lobule is made up of millions of hepatic cells

which are the basic metabolic cells lobules are the

functional units of the liver (Kmiec, 2001).

DIAGRAM OF THE LIVER

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Labels:(1) Right lobe, (2) Left lobe, (3) Caudate Lobe, (4) Quadrate lobe, (5) Hepatic artery and portal vein, (6) Hepatic Lymph Nodes, (7) Gall Bladder.

2.5 Liver Histology

27

Sheets of connective tissue divide the liver

into thousands of small units called lobules. A

lobule is roughly hexagonal in shape, with portal

triads at the vertices and a central vein in the

middle.

The parenchyma cells of the liver are

hepacocytes. These polygonal cells are jointed to

one another in anatomosing plate, with borders that

face either the sinusoids or adjacent hepatocyte.

The ultra structure appearance of hepatocyte

reflects their function as metabolic superstars

with abundant rough and smooth endoplasmic

reticulum and Golgi membrane. Hepatocyte make

contact with blood in sinusoids, which are

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distensible vascular channels lined with highly

frustrated endothelial cells and populated with

phagocytic kepffer cells. The space between

endothelium and hepatocytes is called the space of

disc which collects lymph for delivery to lymphatic

capillaries (Smithuis Robin, 2014).Radiology

retrieved.

2.6 Functions of the Liver

The liver break down or modifies toxic

substance (e.g. methylation) and most medicinal

products in a process called drug metabolism.

This sometimes results in toxication, when the

metabolite is more toxic than its precursor.

The liver converts ammonia to urea.

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The liver glucoronidate bilirubin, facilitating

its excretion into bile.

The liver synthesizes angiotensinogen, a

hormone that is responsible for raising the

blood pressure when activated by rennin, an

enzyme that is released when the kidney senses

low blood pressure.

The livers tore a multitude of substances,

including glucose (in form of glycogen) vitamin

A, Vitamin D, Vitamin B12 Vitamin K, Iron and

Copper.

The produce Albumin , the major osmolar

component of blood serum (Romer Alfred

Sherwood; Parson Thomas, 1997).

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2.6 Relation to Medicine and Pharmacology

The oxidative capacity of the liver disease

with aging and therefore any medication that

require oxidation (for instance, benzodiazepines)

are more likely to accumulate to toxic levels.

However, medication with shorter half-lives such as

lorazepam and oxazepam, are preferred in most cases

when benzodiazepines are required in regards to

geriatric medicine (the branch of medicine

concerned with the diagnosis and treatment of

disorders that occur in old age).

2.7 Diseases of the Liver

The liver supports almost every organ in the

body and is vital for survival. Because of its

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strategic location and multidimensional functions,

the liver is also prone to many diseases.

The most common include; infectious such as

hepatitis A,B,C,D,E, alcohol damage, fatty liver,

cirrhosis, cancer, drug damage (particularly by

acetaminophen [Paracetamol] and cancer drug). Many

diseases of the liver are accompanied by jaundice

caused by increased levels of bilirubin in the

system. The bilirubin results from breakup of

haemoglobin of dead red blood cells. There are also

many pediatric liver disease including biliary

atresia, alpha-l, antitrypsin deficiency, alagille

syndrome, progressive familiar intrahepatic

cholestasis, and langerhans histocytosis, to name

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but a few. Liver diseases may be diagnosed by liver

function test. For example, by production of acute

phase protein (Robbins, Stanley, L. Abbas, Abulka,

2005).

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REFERENCES

Adisa, R.A., Choudharyb, M.I. and Olorunsogo, O.O.(2010). Hypoglycemic activity of Buccholziacoriacea (Capparaceae) seeds in streptozotocin-induced diabetic rats and mice. Elsevier Gmbh,Exp. Toicol. Pathol., 63(7-8); 619-25.

Amaechi, N.C. (2009). Nutritive and Anti-NutritiveEvaluation of Wonderful cola (Buccholzia coriacea)Seeds. Pakistan Journal of Nutrition, 8:1120-112.

Ayayeoba, E.O., Onocha, P.A. Nwozo, S.O., andSama, W. (2003). Antimicrobial and cytotoxicityevaluation of Buccholzia coriacea stem bark.Fitoterapia. 2003; 74 (7-8): 706-709.

34

Burkill, H.M. )2002). The useful plants of westtropical Africa. Royal Botanic Gardens, Kew,319.7. Walker, A.R. Usages pharmaceutiques desplantes spontanus du Gabon, vol. 9 Institutd’Etudes centra Fracaines, 8:13-26.

Ernst, E. (2005). The efficacy of herbal medicine-an overview. Fundamclin. Pharmacol, 19: 405-409.

Ezekiel, O.O. and Onyeoziri, N.F. (2009).Preliminary studies on the antimicrobialproperties of Buccholzia coriacea (wonderful cola).African Journal of Biotechnology, 8(3):472-474.

Hoffman, C. (2003). Comparative antimycotic effectsof selected herbs, spices, plants componentsand commercial antifungal agents. J. Food. Prot.,45:1298-1301.

Jorns, A., Tiedge, M., Lenzen, S. and Munday, R.(1999). Effect of superoxide dismutase,catalase, chelating agents, and free radicalscavengers on the toxicity of alloxan toisolated pancreatic islets in vitro. Free Radic BiolMed., 26:1300-1304.

35

Lenzen, S. (2008). The mechanisms of alloxan-andstreptozotocin-induced diabetes. Diabetologia,51:216-226.

Mbata, T.I., Duru, C.M. and Onwumelu, H.A. (2009).Antibacterial activity of crude seed extract ofBuccholzia coriacea on some pathogenic bacterials.Journal of Developmental Biology and Tissue Engineering,1(1):1-5.

Nweze, N.E. and Asuzu, I.U. (2009). Theantihelminthic effects of Buccholzia coriacea seed.Nig. Vet. J., 27:60-65.

Nweze, N.E., Fakae, L.B. andM Asuzu, I.U. (2008).Anti-trypanosomal activity of the ethanolicextract of Buccholzia coriacea seeds. WOCMAP IVBook of Abstracts, Cape Town. South Africa,103.

Rao, M.U., Sreenivasulu, M., Chengaiah, B.,Reddy,K.J. and Chetty, C.M. (2010). “Herbal Medicinesfor Diabetes Mellitus: A Review,” pp. 921-927.

Tiegde, M., Lortz, S., Drinkgern, J., and Lenzen,S. (1997). Relation between antioxidant enzymegene expression and anti-oxidative defense

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status of insulin-producing cells. Diabetes,46:1733-1742.

Witnerbourn, C.C. and Munday, R. (1989).Glutathione-mediated redox cycling of alloxan.Mechanisms of superoxide dismutase inhibitionand of metal-catalyzed OH formation. BiochemPharmacol. 38:271-277.

Zhang, X., Liang, W., Mao, Y., Li, H., Yang, Y. andTan, H. (2009). Hepatic glucokinase activity isthe primaryd effect in alloxan-induced diabetesofmice. Biomed Pharmacometer. 63:180-186.

APPENDIX I

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Chloroform

Lithium heparin

Cotton wool

What filter paper

Wonder kola

Alloxan

Cage

Glibenclamide (5mg)

Plates

Spectrophotometer

Centrifuge

Glucometer machine

Water bath

Hand gloves

Syringes

Cannula

Reagent

Weighing balance

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APPENDIX II

EXTRACT AND DRUG CALCULATION

4gram of the extract of wonderful kola was weight

with electronic balance and later dissolves in 80ml

of water. This gives a stock solution of

4,000mg/80ml and a concentration of 50mg/ml.

Dosage (mmg) required for a rat of 100g taking a

standard dose of 200mg/kg can be calculated as

follows:

(W) x 200mg

1000g

Where w = body weight of animals

Where 200mg is the standard dose

Dose in mg (150g rat) x 200mg

30,000mg = 30mg

1000g

Equivalent volume required for 30mg dose for 150g

rat was calculated as follows:

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If the concentration (i.e.) 50mg = 1ml

Therefore volume for 30mg = 1ml x 30mg

50mg

= 0.6ml

Drug Calculation

10 tablets of Gliberclamide = 50mg

Distilled water – 20ml

Concentration = 50mg = 2.5mg/ml

20ml

Standard Dose = 5mg

Dosage = Weight x Standard

1000g

Conc.

For experimental animals in group 1 of weight 70g:

Dosage = 70 x 5

1000

2.5

0.14mls

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FOR MORE INFO ON HOW TO GO ABOUT THIS RESEARCH WORK

CONTACT: OGHENEKARO EMMY TEDHEKE ON 08103629140

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