EFFECT OF ETHANOL LEAF EXTRACT OF ANDROGRAPHIS PANICULATA ON

KIDNEY FUNCTION PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS

BY
UDOGU OGECHUKWU BIBIAN

A PROJECT PROPOSAL SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY/BIOCHEMISTRY, SCHOOL OF INDUSTRIAL AND APPLIED
SCIENCE, FEDERAL POLYTECHNIC NEKEDE, OWERRI, IMO STATE

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF

HIGHER NATIONAL DIPLOMA (HND) IN MICROBIOLOGY/BIOCHEMISTRY

SUPERVISOR: AMARALAM ERIE CHINEDU

MAY, 2026
 CHAPTER ONE

INTRODUCTION

1.1 Background of the Stud

Medicinal plants have continued to play a vital role in the management of various diseases dur to

their rich phytochemical composition and therapeutic properties, which have been explored

extensively in modern pharmacological research for the development of safer and more

affordable treatment options (Reddy et al., 2022).

Among these medicinal plants, Andrographis paniculata has gained significant attention for its

diverse biological activities including anti-inflammatory, anti-oxidant, antimicrobial, and

antidiabetic effect, largely attributed to its major bioactive compound, andrograplolide (Ma et al.,

2023).

The ethanol extraction method is widely employed in phytochemical studies because it

effectively isolates active constituents from plant materials, truly enhancing the biological

efficacy of plant extracts used in experimental and therapeutic application (Arozal et al., 2025).

Experimental animal models are essential tools in biomedical research, particularly for

investigating the physiological and biochemical effects of plant extract on organ system, with

rats being commonly used due to their genetic and metabolic similarities to human (Uselu et at.,

2024).

One of the commonly used methods for inducing experimental diabetes in laboratory animals is

through the administration of alloxan, a compound known to selectively destroy pancreatic beta
cells, thereby creating a model suitable for studying metabolic disorders and associated

complications (Nnodim et al., 2024).

The kidney plays a crucial role in maintaining homeostasis by regulating fluid balance,

electrolyte concentration, and the excretion of metabolic waste products making it highly

susceptible to damage under pathological conditions (Arozal et al., 2025).

Alterations in kidney function are commonly assessed using biological parameters such as urea,

creatinine and electrolyte, which serve as reliable indicators of renal health and functional

integrity (Nnodim et at., 2024). Under diabetic conditions, prolonged metabolic imbalance and

oxidative stress can impair renal function, leading to elevated levels of these parameters and

eventual development of nephropathy (Reddy et al., 2022).

Natural products with antioxidant and anti-inflammatory properties have been reported to offer

protective effects against such renal damage by reducing oxidative stress and improving

biological alterations associated with kidney dysfunction (Ma et al., 2023)

Previous investigation on Andrographis paniculata have largely focused on its glucose-lowering

and lipid-modulating activities, with comparatively fewer studies examining its effects on renal

function parameters in experimental diabetic models (Uselu et al., 2024).

Furthermore, the potential of ethanol leaf-extract of Andrographis paniculata to ameliorate

kidney dysfunction in alloxan-induced diabetic rat remains insufficiently, explored, thereby

necessitating further scientific investigation (Arozal et al., 2025).

1.2 Statement of Problem

Several studies have reported the therapeutic potential of Andrographis paniculata, particularly

its antihyperglycemic and antioxidant effect in experimental model. However, despite these

documented benefits, there is limited research specifically addressing the impact of its ethanol

leaf extract on kidney function parameters such as serum urea, creatinine, and electrolyte level in

alloxan-induced diabetic rats (Nnodim et al., models). This gap in existing knowledge highlights

the need for further investigation into its possible nephroprotective effects which this study aims

to explore.

1.3 Aim of the Study

The aim of this study is to evaluate the effects of ethanol leaf extract of Andrographis paniculata

on selected kidney parameters in alloxan-induced diabetic rats.

1.4 Objectives of the Study

The objectives are to:

i. Determine the effect of ethanol leaf extract of Andrographis paniculata on serum urea

levels in diabetic rats.

ii. Evaluate the effect of the extract on serum creatinine levels.

iii. Assess the effect of the extract on electrolyte levels (sodium, potassium, chloride).

1.5 Significance of the Study

This study will provide valuable scientific evidence on the potential nephroprotective effect of

ethanol leaf extract of Andrographis paniculata thereby contributing to the growing body of

knowledge on plant-based therapies for managing complication associated with metabolic

disorders. It may also support the development of affordable and accessible treatment

alternatives, particularly in resource-limited setting where conventional drugs may not be readily
available. Additionally, the finding from this study may serve as a basis for further research and

possible clinical application of Andrographis paniculata in the management if kidney dysfunction

associated with diabetes.

1.6 Scope of the Study

This study will be limited to the following areas:

i. The study is limited to experimental rats induced with alloxan

ii. Only ethanol lea extract of Andrographis paniculata is investigated

iii. The study focuses on kidney function parameters (urea, creatinine, electrolytes).

iv. It excludes human clinical trials.

v. The duration is restricted to the experimental period

 CHAPTER TWO

LITERATURE REVIEW

2.1 Diabetes mellitus

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia

resulting from defects in insulin secretion, insulin action, or both. It is associated with long-term

damage to various organs particularly the kidneys, leading to diabetes nephropathy. Chronic

hyperglycemia induces oxidative stress, inflammation and microvascular damage, which

progressively impair kidney function. One of the earliest manifestation of diabetic nephropathy

is electrolyte imbalance including alteration in sodium (Na +), potassium (K+ ), chlorine (Cl-) and

bicarbonate (HCo). These changes occur due reduced glomerular filtration rate (uf 12) and

tubular dysfunction.

2.2 Alloxan-induced Diabetes in Experimental Models

Alloxan is a widely used diabetogenic agent in experimental research. It selectively destroyed

pancreatic cells through the generation of Reactive Oxygen Species (ROS) leading to insulin

efficiency and hyperglycemia.

Alloxan-induced diabetes mimics type 1 diabetes and is commonly used in studies investigating

antidiabetic agents and their effects on organ system including the kidney. Elevated blood

glucose following alloxan administration results in:

 Increased oxidative stress

 Altered renal hemodynamics

 Electrolyte imbalance
  Kidney tissue damage

 Kidney tissue damage

Studies have shown that alloxan significantly increases blood glucose levels and disrupts

metabolic hemostasis in experimental animals.

2.3 Serum Electrolyte and Kidney Function in Diabetes

The kidney plays a crucial role in maintaining electrolyte balance. In diabetic conditions,

impaired kidney function leads to abnormalities such as:

 Hypernatremia (low sodium) due to osmotic diosmosis

 Hyperkalemia (high potassium) due to reduced-excretion

 Metabolic acidosis (low bicarbonate)

 Altered chloride level

Electrolyte imbalance in diabetes is include to:

 Glomerular damage

 Tubular dysfunction

 Reduced renal clearance

Persistent electrolyte disturbances can worsen disease progression and increase mortality.

2.4 Medicinal Plants in Diabetes Management

Medicinal plants have gained attention as alternative therapies for diabetes due to their

affordability, accessibility, and minimal side effects. Many plant extracts exhibit:

 Antioxidant activity

 Hypoglycemic effect
  Thenprotective properties

These effects are mainly attributed to phytochemicals such as flavonoid, alkaloids, terpenoids,

and glycoside.

2.5 Botanical Profile of Andrographis paniculata

Andrographis paniculata (family Acanthacene) commonly known as “King of Bitters”, is widely

used in traditional medicine across Asia and Africa. It is known for its therapeutic effects in

treating:

 Diabetes

 Inflammation

 Liver disorders

 Infections

The plant contains bioactive compounds such as:

 Andrographolide (major active constituent)

 Flavonoids

 Diterpenes

 Polyphenols

These compounds are responsible for its pharmacological activities

2.6 Ethanol Leaf Extract of Andrographis paniculata

Ethanol extraction is commonly used because it efficiently extracts both polar an non-polar

phytochemicals. Ethanol leaf extract of A. paniculata have been shown to contain higher number

of aqueous extracts. These extracts exhibit:

  Strong anti-oxidant activity

 Anti-inflammatory effects

 Antidiabetic properties

2.7 Antidiabetic effects of Andrographis paniculata

Several studies have demonstrated the hypoglycemic effect of A. paniculata in experimental

animals.

 Ethanol extract significantly reduced blood glucose levels in alloxan-induced diabetic rats

by up to 45%.

 Prolonged administration improved insulin levels and glucose metabolism

These effects suggests that A. paniculata can restore glucose homeostasis and increase pancreatic

function

2.8 Effects on Kidney Function Parameters

Although many studies focus on blood, glucose, emerging research indicates that A. paniculata

also has renoprotective effects.

Experimental studies have shown that ethanol leaf extract reduces oxidative stress in kidney

tisanes, improve renal biochemical parameters, and prevent structural damage to nephrons.

In alloxan-induced diabetic rats, treatment with ethanol extract improved renal biochemical

indices and metabolic parameters, indicating proactive effects on organ function.

2.9 Effects on Serum Electrolytes

Electrolyte imbalance is a major complication of diabetes plant extract with antioxidant and

nephroprotective properties can help restore electrolyte balance.

The mechanism through which A. paniculata may regulate electrolyte includes:

 Improved kidney filtration- enhances GFR and electrolyte reabsorption.

 Antioxidant activity: Reduces Thos-induced renal damage

 Although direct studies on serum electrolytes are involved improvement in kidney

function indirectly stabilizes electrolyte levels.

2.10 Mechanism of Action

The pharmacological effects of ethanol leaf extract A. paniculata are attributed to:

1. Anti-oxidant mechanism

 Scavenges free radicals

 Reduces oxidative stress in renal tissues

2. Insulin mechanism

 Enhances insulin secretion

 Improves insulin sensitivity

3. Enzyme regulation

 Inhibits gluconeogenesis

 Stimulates glycolysis

4. Anti-inflammatory Action

 Reduces cytolaine-mediated kidney damage

5. Nephroprotective Effect

 Prevent structural damage to glomenli and tubules

2.11 Research Gap

Despite extensive research in the antidiabetic properties of A. paniculata there is:

  Limited data on serum electrolyte modulation

 Insufficient studies linking renal function parameters with electrolyte balance

 Need for more dose-dependent and mechanistic study.

This creates gap for studies investigating the effect of ethanol leaf extract of A. paniculata on

kidney function and serum electrolytes in alloxan-induced diabetes rats.

2.2 Review of Previous Research on A. Paniculata

Several researchers have investigated the pharmacological effects of Andrographis paniculata,

particularly its antidiabetic oxidants, and renoprotective properties using experimental animal

models.

2.2.1Antidiabetic studies using Alloxan-induced Diabetic rats

A study by Hossain et al., (2007) evaluated the antidiabetic activity of aqueous and ethanol

extracts of Andrographis paniculata in alloxan-induced diabetic rats. The findings revealed that:

 Ethanol extract reduced blood glucose levels by about 45%.

 Aqueous extract showed similar hypoglycemic effect

 The plant demonstrated strong potential as a natural antidiabetic agent.

This study confined that A. paniculata possesses significant glucose- lowering activity in animal

by induced diabetes models.

Similarly, Yusuf et al., (2022) investigated the effect of aqueous leaf and stem extract of A.

paniculata on alloxan-reduced diabetic Wistar rats. Their findings showed:

 Significant reduction in blood glucose levels.

 Improvement in diabetic condition after extract administration

  Support for the use of the plant in traditional diabetes management.

This study further validated the plants antidiabetic efficacy in experimental animals.

2.2.2 Studies on Antioxidant and Organ-protective Effects

Research by Ogulana et al., (2021) examined the effects of Andrographis paniculata on

streptozotocin-induced diabetic rats. The study reported:

 Significant reduction in blood glucose levels

 Improvement in kidney function parameters

 Change in biochemical molars such as potassium and phosphorus

The authors concluded that A. paniculata ha both diabetic an organ-protective (including renal)

effects especially at higher doses.

2.2.3 Studies on Metabolic and Biochemical Effects

A study by Abbtar et al., (2016) investigated metabolic changes induced by A. paniculata extract

in diabetic rats.

The result showed:

 Improved glucose metabolism

 Reduction in hyperglycemia

 Regulation of metabolic pathways associated with diabetes

The study highlighted the plant’s ability to modulate biochemical processes linked to diabetic

complication.
2.2.4 Studies on Phytochemical and Active Compounds

Recent research has focused on isolating active compound responsible for the plant’s medicinal

properties.

For example:

Bolashi et al., (2023) identified bioactive fraction and compounds in A. paniculata responsible

for its antidiabetic effects.

The major compound, andrographolide was found to play large role in glucose regulation.

These findings support the pharmacological basis of the plant’s therapeutic effects.

2.2.5 A Clinical and a Combined Extract Studies

A clinical-related study by Elya et al., (2018) evaluated a combination of Andrographis

paniculata with other medicinal plants in diabetic patients.

The study showed:

 Improved glycemic-control when used alongside standard drugs like metformin

 Confirmation of its effectiveness beyond animal models

This suggests that A. paniculata may have translational potential for human use

2.2.5 Identified Gap in Literature

Despite numerous studies;

 Few studies have specially focused on serum electrolyte (Na+, K+, Cl-, HCo3-)

 Limited research directly links kidney function parameters with electrolyte balance
 There is insufficient work on ethanol leaf extract on renal electrolyte in alloxan-induced

diabetic
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Equipment/Apparatus

1. Animal cage

2. Calulometer and strips

3. Dissecting set

4. Oral cannula coral gavage

5. Electronic weighing balance

6. Light microscope

7. Syringes, rotary evaporator

8. Refrigerator

9. Mechanical grinder

10. Standard rat feeds

3.2 Chemicals/Reagents

Ethanol (70% or absolute ethanol free extraction) formation (10% neutral buffered formalin for

fixation), Alloxan monohydrate (for diabetes induction), chloroform, whatman No 1 filter paper,

paraffin wax, hematoxylin and cosin stains, standard antidiabetic drug (gliben chloride).

Plant

Leaves of endrographis paniculata

Rats (male and female)

3.3 Methods

3.3.1 Plant Collection and Extraction

Fresh leaves of endrographis paniculata will be collected from a vegetative garden at Umuerim

Nekede, Owerri, Imo State and will be identified by a botanist.

3.3.2 Sample Preparation

The leave of Andrographis paniculata will be air dried under shade for 14 days. The dried ;eaves

will be pulverized into coarse powder. 500g of the powder will be macerated in rust of 70%

ethanol for 72 hours with intermittent shaking. The mixture will be filtered using whatman No 1

filter paper and the filtrate will be concentrated using a rotary evaporator at 40 0c to obtain a

semi-solid extract. The extract will be stored at 40c until use.

3.3.3 Induction of Diabetes

Diabetes will be inducted by a single intraperitoneal injection of alloxan monohydrate at

150mg/kg body weight to overnight fasted rats.

3.3.4 Experimental Design

The rats will be randomly divided into 5 groups of 4 rats each.

GROUPS TREATMENT

A Normal control-receives feed and water only

B 150mg/kg body weight of Alloxan monohydrate

C Induced + standard drug (gliben clamide)

D Induced + 200mg/kg of extract A. paniculata

 Later extract 400

E Induced + 400mg + 800mg/kg extract A. paniculata

All treatment will be administered orally for 14 days.

3.3.5 Blood Sample Collection

At the end of the treatment, all animals will be anaesthetized with diethyl vapour. The animals

will be sacrificed by cervical dislocation. Each of rat will be pegged down on a awing bench and

held firmly with office pin. The blood will be collected into a plain tube. The blood samples will

be collected into a tube. Samples will be centrifuge in a bench top centrifuge (3000rpm for 10

minutes) to obtain serum. The liver tissues will be extracted and preserved in 10% formaldehyde

for histological procedure.

3.4 Peter Mina tun of Kidney Function Parameter

The determination of kidney function parameters refers to laboratory tests used to evaluate how

well the kidney are filtering blood, maintaining electrolyte balance, and excreting waste product.

These parameters are especially important in conditions like diabetes, hypertension, and toxic

exposures.

3.4.1 Method of Determination

i. Colorimetric Method

 Used for creatinine (e.g Jaffe reaction)

 Based on color change measured by spectrophotometer

ii. Enzymatic Methods

 More specific and accurate

 Use for urea, creatinine, and uric acid

Application in Research

(e.g Alloxan-induced diabetic rats)

In studies involving diabetic models, such as alloxan-induced rats, diabetes can cause diabetic

nephropathy (kidney damage)

Researchers measure

i. Creatinine

ii. Urea

iii. Electrolyte imbalance

iv. Plant extracts (e.g Andrographis paniculata) are tested for:

 Nephroprotective effects

 Restoration of normal kidney parameters

3.5 Statistical Analysis

Data will be expressed as mean ± sn. Statistical analysis will be performed using spss (version

20.0). Differences among groups will be analysed using oneway ANOVA followed by Tukey’s

Post hoc test. Values of PL 0.05 will be considered statistically significant.

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