Diuretic Activity of Psidium Guajava Pulp Extract on Albino Rats

There is increasing interest in the health and wellness benefits of herbs and botanicals. Natural medicine is a precious resource of therapeutically active components compounds and has increasingly attracted the attention of researchers; further many studies have reported that herbal diuretics might be a useful tool in the treatment of hypertension and chronic kidney diseases. There are a growing number of studies purporting diuretic effects with traditional medicines.

Diuretics drugs increase the rate of urine flow and adjust the volume and composition of body fluids. Drug-induced diuresis is beneficial for the treatment of many maladies such as congestive heart failure (CHF), chronic renal failure, nephritis, cirrhosis, hypertension and pregnancy-induced toxemia. Medicinal herbs are the significant source as Diuretics. Mono and poly-herbal preparations have been used as diuretics. According to one estimate, more than 650 mono and poly-herbal preparations in the form of decoction, tincture, tablets and capsules from more than 75 plants are in clinical use. The various methods for screening of diuretic agents provides useful tool to evaluate the safety and effectiveness of the drugs. It is also useful for determining the dose level of particular class of diuretic agents. ^{[1 -5]}

High blood pressure represents an important risk factor to development of other cardiovascular diseases and constitutes one of the main causes of mortality in the world.^[6] Diuretic compounds that stimulate the excretion of water are potentially useful in most of disorders including those exhibiting oedema such as congestive heart failure, nephritis, toxemia of pregnancy, premenstrual tension and hypertension.^[7] Diuretic can also increase the elimination of electrolytes.^[8] The modern era of diuretic therapy began in 1949 when sulphanilamide was discovered to possess diuretic and natriuretic properties.^[9] The net excretory effect of diuretic agents causes changes in urine flow, pH, and ionic compositions of urine and blood.^[10]

However, many of the diuretics currently used in clinical practice have been associated with a number of adverse effects, including electrolyte imbalance, metabolic alterations, the onset of diabetes, activation of the renin- angiotensin and neuroendocrine systems, and impairment of sexual function. Therefore, it is important to consider alternatives that have greater effectiveness and fewer side effects. Many of the herbs used in folk medicine have yet to be scientifically evaluated for their effectiveness and safety^[11]

For thousands of years, humans have been using diuretics to reduce the water retention caused by some health conditions such as high blood pressure, heart diseases or pre-menstrual syndrome, among others. Although there is a wide therapeutic stock of synthetic drugs that belong to this pharmacological group, a considerable amount of decoctions and in fusions of medicinal plants are used to reduce fluid retention.But the diuretic effectiveness of this kind of medicinal plants needs to be experimentally proved, because diuresis could be influenced not only by the form of administration which implies the consumption of a great amount of liquids that can provoke an increase in the volume of urine excreted without a true evidence of a diuretic action, but also by the difficulty of obtaining reproducible data involving a larger number of animals.^[11]

Table 1: Classification and adverse effects of diuretics ^[12,13]

Human beings utilize many species of flora for food and medicine. It is also expected that the traditional and modern medicine uses about 50,000 - 70,000 species of plants.^[8] Herbal drugs have gained importance and popularity in recent years because of their safety, efficacy and cost effectiveness.^[4]Medicinal plants can be significant sources of undiscovered chemical substances with potential therapeutic effects. In fact, the World Health Organization has estimated that over 75% of the world’s population still relies on plant-derived medicines, usually obtained from traditional healers, for basic healthcare needs.^[14] Regardless of their specific pharmacological effects, most medicinal plants have been described as having a more or less pronounced diuretic effect. However, very few studies have examined the mechanisms of action of inducing renal excretion.^[6] Some of the diuretics are derived from medicinal plants and a vast number of medicinal plants mentioned in Ayurvedic system of medicine are known to possess diuretic properties such as Abelmoschusesculentus, Bacopamonnieri, Barbara vulgaris and Cissampelospareira.^[15]

Number of species and genuses reporting diuretic effects. of these, the most promising, at the present time, are the speciesFoeniculumvulgare, Fraxinus excelsior, Hibiscus sabdariffa, Petroselinumsativum and Spergulariapurpurea, and species from the genusesCucumis (Cucumismelo and Cucumistrigonus), Equisetum (Equisetum bogotense, Equisetum fluviatile, Equisetum giganteum, Equisetum hiemale var. affine and Equisetum myriochaetum), Lepidium (Lepidiumlatifolium and Lepidiumsativum), Phyllanthus (Phyllanthusamarus,Phyllanthuscorcovadensis and Phyllanthussellowianus) and Sambucus (Sambucusmexicana and Sambucusnigra).

So, apart from this Psidium guajava, Guava is highly nutritious and a good source of calcium, iron, and phosphorus. The vitamin C content of guava fruit is 2–5 times that of citrus. The plant has many medicinal properties mainly due to its bioactive phytoconstituents. This plant finds applications for the treatment of diarrhea, dysentery, gastroenteritis, hypertension, diabetes, caries and pain relief and for improvement in locomotors coordination. Its fruit is rich in vitamins A, C, iron, phosphorus and calcium and minerals. It contains high content of organic and inorganic compounds like secondary metabolites e.g. antioxidants, polyphenols, antiviral compounds, anti-inflammatory compounds. The phenolic compounds in guava help to cure cancerous cells and prevent skin aging before time. The presence of terpenes, caryophyllene oxide and p-selinene produces relaxation effects. Guava leaves contain many compounds which act as fungistatic and bacteriostatic agents. Guava has a high content of important antioxidants and has radio-protective ability.

So, keeping the above all the things in view, it was found that there were no research conducted on the diuretic activity of Psidium guajava and also due to presence of the  antioxidant and polyphenolic constituents, the present study is planned to evaluate the Diuretic potential of Psidium guajava.^[15]

PSIDIUM GUAJAVA L. is common guava, yellow guava, or lemon guava. Psidium guajava is considered a native to Mexico which further developed in South America, European, Africa and Asia. Historically this has been widely used in Peru since pre-Columbian times.^[18] These are characterized by a low content of carbohydrates (13.2 %), fats (0.53 %), and proteins (0.88 %) and by a high water content (84.9 %), Food value per 100 g is: Calories 36–50 kcal, moisture 77–86 g, crude fibre 2.8–5.5 g, ash 0.43–0.7 g, calcium 9.1–17 mg, phosphorus, 17.8–30 mg, iron 0.30–0.70 mg , vitamin A 200–400 I.U., thiamine 0.046 mg, riboflavin 0.03–0.04 mg, niacin 0.6–1.068 mg, ascorbic acid 100 mg, vitamin B3 40 I.U. Manganese is also present in the plant in combination with phosphoric, oxalic and malic acids. Hexanal (65.9 %), butyrolactone (7.6%), (E)-2-hexenal (7.4 %), (E,E)-2,4-hexadienal (2.2%), (Z)-3-hexenal (2 %), (Z)-2-hexenal (1 %), (Z)-3-hexenyl acetate (1.3 %) and phenol (1.6  %) were reported from fresh white-flesh guayaba fruit oil. 3-caryophyllene (24.1 %), nerolidol (17.3%), 3-phenylpropyl acetate (5.3 %) and caryophyllene oxide (5.1 %) were isolated from essential oil extracted from the fruits. Subsequently, the active aromatic constituents in pink guava fruit the 3-penten-2-ol and 2-butenyl acetate were isolated. The fruit also contains glykosen 4.14 %, saccharose 1.62 %, and protein 0.3 %. The unripe fruit is high in tannins, is astringent and has a tendency to cause constipation, but it is sometimes employed in diarrhoea.The main traditional use known is as an anti-diarrhoeal. Other reported uses include gastroenteritis, dysentery, stomach, antibacterial colic pathogenic germs of the intestine. ^[16]

Table 2: Pharmacological Study ^[16]

DIURETICS drugs increase the rate of urine flow and adjust the volume and composition of body fluids. Drug-induced diuresis is beneficial for the treatment of many maladies such as congestive heart failure (CHF), chronic renal failure, nephritis, cirrhosis, hypertension and pregnancy-induced toxemia. ^[17]

FUROSEMIDE ^{[17, 18]}

Furosemide is a loop diuretic used to treat hypertension and edema in congestive heart failure, liver cirrhosis, renal disease, and hypertension. It is an anthranilic acid derivative. Furosemide promotes diuresis by blocking tubular reabsorption of sodium and chloride in the proximal and distal tubules, as well as in the thick ascending loop of Henle. This diuretic effect is achieved through the competitive inhibition of sodium-potassium-chloride co-transporters expressed along these tubules in the nephron, preventing the transport of sodium ions from the lumenal side into the basolateral side for reabsorption. Dose:For edema the usual initial dose of is 20 to 80 mg given as a single dose. The dose may be carefully titrated up to 600 mg/day in patients with clinically severe edematous states. For Hypertension the usual initial dose is 80 mg, usually divided into 40 mg twice a day.

DIURETIC ACTIVITY IN RATS (LIPSCHITZ Test) ^{[19 – 25]}

The Lipschitz test has been proven to be a standard method and a very useful tool for screening of potential diuretics. A method for testing diuretic activity in rats has been described by Lipschitz et al. (1943). The test is based on water and sodium excretion in test animals and compared to rats treated with a high dose of urea. A method using rats for estimation of antidiuretic potency was described by Burn in 1931. This method or a modification of it has been used for diuretic assays by most of the subsequent workers. In 1943, Lipchitz et at. described a method suitable for diuretic assay using several commonly used diuretics. This method by itself is a modification of Burn's method. Since 1943, most of the workers have used the method of Lipschitz with some Modifications.

MATERIALS AND METHODS

ANIMALS:

Albino rats (Wistar) weighing 150-200 g were used in the study. Animals were acquired from Dr. V. V. P. F's College of Pharmacy, Ahmednagar. Animal house and were acclimatized for one week under laboratory conditions. The animals were housed in groups of six in polypropylene cages and maintained at an ambient temperature under 12 hours of light/ dark cycle. They were provided with commercial food pellets and tap water was provided. Ethical clearance for handling the animals was obtained from the institutional animal ethics committee before the beginning of the project work. (1670/PO/ReBiBt/S/12/CPCSEA).

METHOD FOR EVALUATION OF DIURETIC ACTIVITY^{[26, 27]}

The Lipchitz method was employed for the assessment of the Diuretic activity. 24 hours fasted rats were used for the Diuretic activity. The rats were divided into 6 groups of 6 animals each.

Group I served as a control and received normal saline orally (25ml/kg). Group II animals served as Standard and received the same amount of normal saline intraperitoneally in which Furosemide at a dose of 20mg/kg b.w. was dissolved. The other groups received normal saline orally (25ml/kg) in which Psidium guajava pulp extract were dissolved. Group III animals received 200mg/kg p.o.of pulp extract, Group IV animals received 400mg/kg p.o. of pulp extract,

All the drugs were freshly prepared prior to administration. Each of these preparations were given in such manner so that the fluid intake was the same in all cases. After dosing, different groups of animals were placed into different metabolic cages. These metabolic cages are specially designed to separate urine and feces. The volume of urine was collected and measured at the end of the 5 and 24 hours respectively. During this period, no water and food was made available to animals.

ANALYSIS OF URINE:

The amount of concentration of Na +, K+ and cl- in the urine sample were determined with the help of flame photometer.

Computation of Diuretic Parameters^[25]:

a. Diuretic Index: Vt/ Vc

b. Lipschitz Value: Vt/ Vr

c. Saluretic Index: Ct/Cc

d. Na+/ K+ ratio :Cn/ Ck

Where,

Vt : urine volume of test group.

Vc : urine volume of control group.

Vr : urine volume of the reference group.

Ct  : concentration of electrolyte in urine of test group.

Cc  : concentration of electrolyte in urine of control group.

Cn : concentration of sodium ion in urine of group &

Ck : concentration of potassium in urine of same group

STATISTICAL ANALYSIS:

Arithmetic mean of the values of readings wear calculated for each experiment the result obtained was used for statistical analysis using INTA software. The data obtained from various models of diuretic activity of rats’ experiments were subjected to analysis of variance (ANOVA) followed by Dunnett’-test using INTA software. The value of p<0.001 was considered statistically significant

RESULTS

EVALUATION OF DIURETIC ACTIVITY  (LIPSCHITZ MODEL)

The diuretic activity was assessed by determination of Lipschitz value using the standard diuretic drug Furosemide. A significant increase in the urinary excretion of electrolytes (Na⁺ and K⁺) over 24 hours in furosemide treated group. Furosemide treated group showed maximum diuretic effect (diuretic index 2.53; Lipschitz value 2.05) lasting over 24 h.

Animals were divided in total of three groups (n = 6 in each group). All animals were deprived of food and water 18 h prior to the experiment. On the day of experiment, the dosing was scheduled as follows.

Immediately after the dosing, animals were placed in metabolic cages and urine was collected up to 5 hours and 24 hours after dosing. Room temperature was maintained up to 25± 0.5 ⁰C.During this period no water or food was made available to the animals.

Table 3: Urinary excretion data

N = 6, Mean ± S.E.M. Significant, *** P < 0.001 Vs. Control

Table 4 Urinary excretion of electrolytes (5 h)

Values are the mean ± S.E.M. of six rats / treatment. Significant **P < 0.05, *** P < 0.001 Vs. Control.

Table 5: Urinary excretion of electrolytes (24 h)

 Values are the mean ± S.E.M. of six rats / treatment. Significant ** P < 0.001 Vs. Control.