Evalution Of Nephroprotective Activity Of Papaya [Carica Papaya] Seed Extract Against Gentamicin-Induced Nephrotoxicity In Albino Wistar Rats

Nephrotoxicity is defined as the adverse effect of exogenous substances such as drugs, chemicals, heavy metals, and environmental toxins on the structure and functional integrity of the kidneys. The kidneys are highly specialized organs responsible for filtration of blood, excretion of metabolic waste products, maintenance of electrolyte and acid–base balance, regulation of fluid volume, and secretion of hormones such as renin and erythropoietin. Because they receive nearly 20–25% of the cardiac output and actively concentrate substances during urine formation, the kidneys are particularly susceptible to toxic injury.

Renal toxicity can involve different anatomical components of the kidney, including the glomeruli, proximal and distal tubules, loop of Henle, collecting ducts, interstitium, and renal vasculature. Among these, the proximal tubules are most commonly affected due to their high metabolic activity and role in reabsorption and secretion. Mechanisms of nephrotoxicity include direct cytotoxic effects on tubular epithelial cells, oxidative stress due to excessive production of reactive oxygen species (ROS), mitochondrial dysfunction, inflammation, immune-mediated injury, and intratubular crystal precipitation leading to obstruction. Reduced renal blood flow and ischemia further aggravate renal damage. Clinically, nephrotoxicity often manifests as acute kidney injury (AKI), characterized by a rapid decline in glomerular filtration rate (GFR), elevated serum creatinine and blood urea nitrogen (BUN), electrolyte disturbances, and decreased urine output (oliguria). If the insult persists or is repeated, it may lead to chronic kidney disease (CKD), progressive nephron loss, interstitial fibrosis, and eventually end-stage renal disease (ESRD).

Common nephrotoxic agents include antibiotics (e.g., aminoglycosides), antiviral drugs such as Acyclovir, non-steroidal anti-inflammatory drugs (NSAIDs), chemotherapeutic agents like cisplatin, radiographic contrast media, and heavy metals. The severity of nephrotoxicity depends on factors such as dose, duration of exposure, route of administration, drug interactions, hydration status, age, genetic susceptibility, and pre-existing renal impairment.

Early detection of nephrotoxicity is crucial for preventing irreversible damage. Traditional biomarkers include serum creatinine, blood urea nitrogen, and creatinine clearance. Recently, novel biomarkers such as kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and N-acetyl-β-D-glucosaminidase (NAG) have shown promise for early identification of renal injury before significant functional decline occurs.

Understanding the pathophysiology, risk factors, and diagnostic approaches of nephrotoxicity is essential for improving drug safety, minimizing renal complications, and developing protective therapeutic strategies.

STANDARD DRUG ACYCLOVIR

Acyclovir is a synthetic antiviral medication widely used in the treatment of infections caused by herpes viruses, particularly herpes simplex virus type-1 (HSV-1), herpes simplex virus type-2 (HSV-2), and varicella-zoster virus (VZV). It is a guanine nucleoside analog that selectively inhibits viral DNA synthesis, thereby preventing viral replication.

The antiviral action of acyclovir depends on its selective activation within infected cells. Initially, it is phosphorylated by viral thymidine kinase to form acyclovir monophosphate. Host cellular enzymes then convert it into acyclovir triphosphate, the active form. This active metabolite competitively inhibits viral DNA polymerase and incorporates into viral DNA, causing premature chain termination. Due to this selective mechanism, acyclovir has a high therapeutic index and minimal toxicity to normal host cells.

Pharmacokinetically, acyclovir has relatively low oral bioavailability and limited hepatic metabolism. It is primarily eliminated unchanged through the kidneys by glomerular filtration and active tubular secretion. Because of its renal excretion, high intratubular concentrations may occur, especially at high doses or with inadequate hydration.

Clinically, acyclovir is available in oral, topical, and intravenous formulations and is commonly prescribed for conditions such as genital herpes, cold sores, herpes encephalitis, and shingles. Although generally well tolerated, adverse effects may include gastrointestinal disturbances, headache, and, in some cases, nephrotoxicity, particularly with high-dose intravenous administration. Overall, acyclovir remains one of the most effective and widely used antiviral agents for herpes virus infections.

Fig.01: Acyclovir dispersible  I.P.400 mg tablets

TEST DRUG  PAPAYA SEEDS

Papaya seeds obtained from the fruits of Carica papaya have attracted considerable attention in recent years due to their diverse medicinal properties. Traditionally regarded as agricultural waste, papaya seeds are now recognized as a rich source of bioactive phytoconstituents with significant pharmacological potential. The seeds contain alkaloids such as carpaine, flavonoids, phenolic compounds, tannins, saponins, fixed oils, fatty acids, and benzyl isothiocyanate, which contribute to their therapeutic effects.

Among their various biological activities, papaya seeds are well known for their antioxidant and anti-inflammatory properties. These properties are particularly important in conditions involving oxidative stress and cellular injury, such as drug-induced nephrotoxicity. The antioxidant components help neutralize reactive oxygen species (ROS), reduce lipid peroxidation, and protect renal tubular cells from damage. Additionally, papaya seeds have been reported to enhance endogenous antioxidant enzymes like superoxide dismutase, catalase, and glutathione, thereby strengthening the body’s natural defense system.Experimental studies have also demonstrated nephroprotective potential of papaya seeds by improving renal biochemical markers such as serum creatinine and blood urea nitrogen, and by preserving normal kidney architecture in toxic models. Their natural origin, availability, cost-effectiveness, and relatively safe profile at therapeutic doses further support their selection as a test drug in experimental research.Therefore, due to their rich phytochemical composition and promising renoprotective properties, papaya seeds were selected as the test drug to evaluate their protective effect against drug-induced nephrotoxicity

Papaya seed powder from Carica papaya was selected because it has strong antioxidant and antiinflammatory properties. Acyclovir causes kidney damage mainly by producing harmful free radicals (oxidative stress), inflammation, and injury to kidney tubules. Papaya seeds contain natural compounds like flavonoids and phenols that help neutralize free radicals, reduce inflammation, and protect kidney cells from damage. Studies suggest that papaya seeds can improve kidney function by lowering serum creatinine and blood urea levels and by supporting natural antioxidant enzymes in the body. They are also easily available, cost-effective, and generally safe at proper doses. Therefore, papaya seed powder was considered a suitable natural option to study its protective effect against acyclovir-induced nephrotoxicity.

Fig.02: Papaya seeds

DRUG PROFILE

 ▪ Common Name: Papaya Seeds

▪ Botanical Name: Carica papaya

▪ Family: Caricaceae

▪ Synonyms: Pawpaw seeds, Papaw seeds ▪ Part Used: Seeds

▪ Geographical Distribution: Cultivated extensively in tropical and subtropical regions including India, Southeast Asia, Africa, and South America.

▪ Major Chemical Constituents: Alkaloids (carpaine), flavonoids, phenolic compounds, saponins, tannins, fixed oils, fatty acids, and benzyl isothiocyanate.

▪ Pharmacological Activities: Exhibits antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, antidiabetic, and nephroprotective properties.

▪ Therapeutic Applications: Used in the management of kidney disorders (renoprotective role), liver ailments, digestive disturbances, parasitic infections, and certain metabolic disorders.

Fig.03: Papaya seed powder

MATERIALS AND METHODS

MATERIALS: -

Plant and parts used:

Papaya seeds (Carica papaya) show nephroprotective activity due to their antioxidant and anti- inflammatory phytochemicals, which reduce oxidative stress, protect renal cells, and help maintain normal kidney structure and functions

Chemicals and Reagents:-

Ferric chloride is used to detect phenols, lead acetate for toxicity studies, chloroform as an extraction solvent, concentrated sulfuric acid and hydrochloric acid for qualitative tests, magnesium ribbon for flavonoid detection, and sodium hydroxide for phytochemical analysis.

Standard Drug: -

Acyclovir is an antiviral drug used mainly in the treatment of herpes simplex virus and varicella- zoster virus infections. It works by inhibiting viral DNA synthesis, thereby preventing the replication of the virus and reducing the severity and duration of infection.

Test Animal: -

Albino rats (Rattus norvegicus) were used in the study after taking prior approval from the Institutional Animal Ethics Committee, following standard national guidelines for the care and use of laboratory animals. 

Animal care and handling procedures were carried out in accordance with CPCSEA guidelines and received approval from IAEC (Approval number:2375\PO\Re\s\2025\CCSEA). In this study, thirty healthy female Albino Wistar rats weighing between 200 and 250 g are taken. The rats were housed in polypropylene cages and they were subjected to a one-week acclimatization period. During this period, they had to adapt to a new environment while adhering to standard husbandry conditions, including a (12:12 hour) light & dark cycle, humidity (30 -70%), temperature (23±2°C), and unrestricted access to their daily dietary requirements of standard pellet diet and water, all of which were provided without inducing stress.

METHODS:-

Ripe fruits of Carica papaya were collected from a local market, and the seeds were carefully separated from the pulp. The seeds were washed thoroughly with distilled water to remove adhering mucilage and impurities and then shade-dried at room temperature for about 7–10 days until completely dry. The dried seeds were powdered using a grinder and stored in an airtight container.

Extraction:-

One hundred grams of the papaya seed powder were taken in a conical flask and extracted with 1000 mL of distilled water by hot maceration. The mixture was heated on a water bath at 60– 70 °C for 2–3 hours with occasional stirring, allowed to cool, and then filtered using Whatman No.1 filter paper. The filtrate was concentrated on a water bath to obtain a semi-solid mass, which was dried and stored in an airtight container at 4 °C for further experimental use.

Test solution:-

The extract was mixed with distilled water to make 200 and 400 mg/kg solution.

TEST DRUG EXTRACT (PAPAYA SEED)

• About 100 g of dried seeds were coarsely powdered.
• Dried papaya seeds were collected, cleaned, and shade dried properly.
• The powder was passed through sieve No. 60 to obtain uniform particle size.
• The sieved powder was transferred into a clean round-bottom flask.
• 1000 mL of distilled water was added as the extraction solvent.
• The mixture was heated at 60 °C for 2 hours with continuous stirring for aqueous extraction.
• After heating, the mixture was allowed to cool to room temperature.
• The extract was filtered using Whatman No. 1 filter paper to remove solid residues.
• The filtrate was concentrated on a water bath until a semisolid mass was obtained.
• The concentrated extract was collected, stored in an airtight container, and preserved at 4 °C for further experimental use.

Fig.04 : Papaya seed Extraction

CHEMICAL TESTS:

1. DRAGENDORFF’S TEST

Papaya seed extract

Add dilute hydrochloric acid and warm gently

Cool and filter

To 2 ml of filtrate add a few drops of Dragendorff’s reagent

Observation: Orange or reddish-brown precipitate

Inference: Alkaloids present

Fig.05: Test for Alkaloids

2. TEST FOR FLAVONOIDS (ALKALINE REAGENT TEST)

A small quantity of the aqueous papaya seed extract was taken to detect the presence of flavonoids.

Papaya seed extract

Add a few drops of sodium hydroxide solution (10%)

Observation: Formation of an intense yellow coloration

Add dilute hydrochloric acid

Observation: Disappearance of yellow color (becomes colorless)

Inference: Presence of flavonoids

Fig.06: Test for Flavonoids

3. TEST FOR TANNINS (FERRIC CHLORIDE TEST)

A small quantity of the aqueous papaya seed extract was taken for the detection of tannins.

Papaya seed extract

Add a few drops of 5% ferric chloride solution

Observation: Formation of blue-black or greenish-black coloration

Inference: Presence of tannins

Fig.07: Test for Tannins

4. TEST FOR TERPENOIDS (SA LKOWSKI TEST)

A small quantity of the aqueous papaya seed extract was taken for the detection of terpenoids.

Papaya seed extract

Add 2 mL of chloroform and mix well

Carefully add 1–2 mL of concentrated sulfuric acid along the side of the test tube

Allow the mixture to stand undisturbed

Observation: Formation of a reddish-brown coloration at the interface

Inference: Presence of terpenoids

Fig.08: Test for Terpenoids

STANDARD DRUG EXTRACT (ACYCLOVIR)

• Acyclovir was selected as the standard drug due to its well-known nephrotoxic potential and clinical relevance.
• The drug was procured from a reputed pharmaceutical source and was of analytical grade.
• The required quantity was accurately weighed daily using a digital analytical balance.
• The dose was calculated at 100 mg/kg body weight for each animal.
• The weighed drug was freshly dissolved in distilled water to obtain the required concentration.
• The solution was mixed thoroughly to ensure uniformity and clarity before administration.
• The prepared solution was administered orally once daily for 14 days using an oral gavage.
• The dose volume was adjusted according to the individual body weight of each animal to ensure accurate dosing.
• Animals were observed throughout the treatment period for any behavioral changes or signs of toxicity.
• Oral administration was chosen to simulate clinical exposure and maintain consistency across treatment groups for proper evaluation of nephrotoxic and nephroprotective effects.

Fig.09: Standard drug Extraction

PROCEDURE 

Healthy albino rats of either sex, weighing about 150–200 g, were obtained from a registered animal house. The animals were housed in clean polypropylene cages under standard laboratory conditions with controlled temperature, humidity, and a 12-hour light– dark cycle. They were provided with standard pellet diet and water ad libitum and allowed to acclimatize before the start of the experiment and divided into five groups control, disease control, standard drug + gentamicin, test drug.

1. Healthy adult Wistar albino rats (150–200 g) were selected and acclimatized for 7 days under standard laboratory conditions with free access to standard pellet diet and water ad libitum.
2. After acclimatization, animals were randomly divided into five groups, each containing six rats (n = 6).
3. The total duration of the experimental study was 14 days.
4. Group I (Normal Control) received normal saline (1 ml/kg, p.o.) once daily for 14 days.
5. Group II (Disease Control) received Gentamycin 10 mg/kg (i.p.) once daily for 14 days to induce nephrotoxicity.
6. Group III (Standard Drug Group) received Gentamycin 10 mg/kg (i.p.) along with Acyclovir 200 mg/kg (p.o.) once daily for 14 days.
7. Group IV (Test Drug – Low Dose) received Gentamycin 10 mg/kg (i.p.) along with Papaya seed powder extract 100 mg/kg (p.o.) once daily for 14 days.
8. Group V (Test Drug – High Dose) received Gentamycin 10 mg/kg (i.p.) along with Papaya seed powder extract 400 mg/kg (p.o.) once daily for 14 days.
9. Body weights were recorded on Day 0 and Day 14 to assess general health status.
10. On Day 15, blood samples were collected under mild anesthesia and serum was separated for estimation of biochemical parameters such as serum creatinine, blood urea, and uric acid.
11. The animals were sacrificed, kidneys were excised, washed with ice-cold normal saline, and preserved in 10% formalin for histopathological examination to evaluate nephroprotective activity.

Fig.10: Protocol