Physico-chemical studies and evaluation of diuretic activity of Cucurbita maxima
Abstract
In this study physico-chemical nature and diuretic activity was evaluated to establish the purity and diuretic activity by comparing with the standard acetazolamide. Pulp of Cucurbita maxima is a common cost effective Indian dish, rich in nutrients. Physico-chemical parameters like ash values, extractive values and loss on drying were performed to find the purity. The hydroalcoholic extract was prepared by extracting the powder in soxhlet apparatus for 36 hours. The laboratory qualitative analysis was done and constituents like carbohydrates, proteins, lipids, flavonoids, alkaloids and vitamin C were found. The two doses of extract (150 and 300 mg/kg) were given to the rats. Group I served as control, Group II served as standard and Group III and IV served as test. Electrolytes and urine volume was measured after 5 hours of treatment and the same was compared with the control and the standard drug acetazolamide. Physico-chemical parameters were in the limit and the extract shows significant (p<0.01) diuretic activity at the dose of 300 mg/kg when compared with control.
Introduction
Medicinal plants can be important sources of unknown chemical substances with potential therapeutic effects. Besides, 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 (Farnsworth et al., 1985). Herbal formulations involve use of fresh or dried plant parts. Correct knowledge of such crude drugs is very important aspect in preparation, safety and efficacy of the herbal product (Modi et al., 2010). In developed countries due to lack of documentation and stringent quality control, herbal drugs were not used much. Correct identification and quality assurance of the starting materials is an essential prerequisite to ensure reproducible quality of herbal medicine which will contribute to its safety and efficacy (Thomas et al., 2008). Standardization is essential measure for quality, purity and sample identification.
Diuretics are drugs that increase the rate of urine flow, sodium excretion and are used to adjust the volume and composition of body fluids in a variety of clinical situations. Drug-induced diuresis is beneficial in many life-threatening disease conditions such as congestive heart failure, nephritic syndrome, cirrhosis, renal failure, hypertension, and pregnancy toxemia. Most diuretic drugs have the adverse effect on quality of life including impotence, fatigue and weakness (Vanamala et al., 2012).
The plant Cucurbita maxima Duchesne (commonly known as pumpkin) belongs to the family of Cucurbitaceae is widely cultivated throughout the world for use as vegetable as well as medicine. Both fruits and the aerial parts are commonly consumed as vegetable. It is a large climbing herb, annual or perennial. Its aerial part consists of flexible succulent stem with trifoliate leaves (Kirtikar et al., 2003). Traditionally it is used in most countries as anti-diabetic, antitumor, antihypertensive, anti-inflammatory, immunomodulatory and antibacterial agents (Caili et al., 2006).
Materials and Methods
Collection of pulp
The pulp of C. maxima was collected along with plant and authenticated in botanical survey of India, Coimbatore. The inner pulp was scrapped and dried in shade.
Determination of ash values (Divakar, 2005)
The ash remaining following the ignition of medicinal plants is determined by three different methods which measures, total ash, acid-insoluble ash, sulfated ash and water soluble ash.
Determination of extractive values
Extractive values are useful to evaluate the nature of constituents present in the crude drug. Water soluble extractive and alcohol soluble extractive values were calculated as per standard procedures.
Determination of moisture content (Loss on drying)
1.5 g of powder was taken in weighed porcelain dish and heated for 100 or 105ºC. cooled and the difference in the weight was calculated as moisture.
Preparation of extract
The coarsely powdered pulp was packed in soxhlet apparatus and boiled for 36 hours with 1:1 ratio of alcohol, water, and then the extract was filtered, concentrated, weighed and stored in desiccators for further studies (Harbone, 1998).
Preliminary phyto–chemical analysis (Sahu et al., 2010)
The hydroalcoholic extract was subjected to the prelimnary phytochemical investigation for the detection of carbohydrates, proteins, lipids, flavonoids, alkaloids, vitamin C and vitamin E (Hancock et al., 2003; Tadmor et al., 2005).
Animals
Twenty four healthy adult albino rats weighing 150-220 g were used. Animals were divided into 4 groups comprised six animals (n = 6). They were housed in standard environmental conditions. The rats were fed with standard diet and water ad libitum. All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics Committee and were in accordance with the Institutional ethical guidelines.
Diuretic activity
Fifteen hours prior to the experiment food and water were withdrawn. Diuretic activity was measured by collecting total excreted urine (0-5 hours) of rat kept in metabolic cage. The cages together with the funnel and measuring cylinder used in the studies were coated with liquid paraffin before each experiment to facilitate the collection of urine with a minimum loss. Each animal was placed in metabolic cage provided with a wire mesh at the bottom and a funnel to collect the urine. Stainless steel sieves were placed in the funnel to retain feces and to allow the urine to pass. Rats were placed in metabolic cages individually as soon as the treatments started. The urine sample was collected for a total period of 5 hours.
Group I: Control group received 5 mL of 0.9% NaCl solution per kg body weight; Group II: Reference control group received acetazolamide (45 mg/kg body weight in 5 mL of 0.9% NaCl solution); Group III: Test group received C. maxima extract (150 mg/kg body weight in 5 mL of 0.9% NaCl solution suspending in 1% carboxy methyl cellulose); Group IV: Test group received C. maxima extract (300 mg/kg body weight in 5 mL of 0.9% NaCl solution suspending in 1% carboxymethyl cellulose).
The total volume of urine in 5 hours after the drug treatment was measured and the urine was subjected to estimation of total concentration of Na+, K+, and Cl-. Na+, K+ concentrations were measured by Flame photometry and Cl- concentration was estimated by titration with silver nitrate solution (N/50) using three drop of 5% potassium chromate solution as indicator (Yar et al., 2009).
Result and Discussion
The physico-chemical values of the powder shows the purity (Table I). Moisture is an inevitable component of crude drugs, which must be eliminated as far as practicable. Deterioration time of the crude drugs depends upon the amount of water present in formulation. If the water content is high, the crude drugs can be easily deteriorated due to fungus and the moisture content of the crude drugs was found to be 10% w/w which signifies that the material was absorb water from the moisture due to the presence of carbohydrate, so it should be properly dried and properly stored. Pulp contains carotenoids and vitamin C (Nawirska-Olszaska and Biesiada, 2011) the compound which can be affected by light so it should be stored without exposing to the sunlight. The total ash value was found to be 16.6% w/w which includes physiological and non-physiological ash. Total ash usually consists of phosphates, carbonates, silicates and silica.
Table I: Physico-chemical parameters of C. maxima powder
| Parameters | Inference |
|---|---|
| Appearance | Powder |
| Colour | Yellowish white |
| Taste | Slight sweetish starchy |
| Odor | Characteristic |
| Total ash | 16.6% w/w |
| Sulphated ash | 26% w/w |
| Water soluble ash | 4% w/w |
| Acid-insoluble ash | 6.5% w/w |
| Water soluble extractive | 5.8% w/w |
| Alcohol soluble extractive | 19.2% w/w |
| Loss on drying | 10% w/w |
Water soluble ash was 4% w/w, sulphated ash was 26% w/w and acid-insoluble ash was 6.5% w/w. More value of sulfated ash indicates the presence oxides and carbonates which are converted into sulfates (Chaudhri, 1996).
Water soluble extractive value and alcohol soluble extractive values were found to be 5.8 and 19.2% w/w respectively indicates the nature and solubility of the chemical constituents.
The diuretic activity of C. maxima was measured in rats by administered the extract in two doses (150 mg/kg and 300 mg/kg). Hydroalcoholic extract of C. maxima at 150 mg/kg showed less significant (p<0.05) diuretic activity and 300 mg/kg showed more significant (p<0.01) diuretic activity when compared with control (Table II).
Table II: Effect of C. maxima extract on urinary output in rats
| Treatment groups | Dose | Total urinary output | Normal saline intake | %Urinary excretion | Diuresis | |
|---|---|---|---|---|---|---|
| Diuretic action | Diuretic activity | |||||
| Control | 1 mL/kg body weight in 5 mL of 0.9% NaCl | 4.7 ± 0.2 | 4.9 ± 0.2 | 95.1 | 1.0 | 0.5 |
| Acetazolamide | 10 mg/kg body weight in 5 mL of 0.9% of NaCl | 9.4 ± 0.2b | 4.8 ± 0.3 | 193.8 | 2.0 | 1.0 |
| C. maxima | 150 mg/kg body weight in 5 mL of 0.9% of NaCl | 6.3 ± 0.3a | 5.0 ± 0.3 | 125.1 | 1.3 | 0.6 |
| C. maxima | 300 mg/kg in 5 mL of 0.9% of NaCl | 9.6 ± 0.3b | 5.0 ± 0.2 | 192.7 | 2.0 | 1.0 |
| Values are in mean ± SEM; ap<0.05 Vs control; bp<0.01 Vs Control; Urinary excretion = Total urinary output /total liquid intake x 100; Diuretic action = Urinary excretion of treated group/urinary excretion of control group; Diuretic activity = Diuretic action of treated group/diuretic action of standard group | ||||||
Diuretic action of the extract at 300 mg/kg was as potent as the standard drug acetazolamide. When compared the electrolyte excretion efficiency of extract with the standard, 300 mg/kg shows more excretion of Na+, Cl– and K+. But 150 mg/kg shows less excretion (Table III).
Table III: Effect of C. maxima extract on urinary electrolytes excretion in rats
| Treatment groups | Dose | Electrolytes( meq/L) | Na+/ K+ | ||
|---|---|---|---|---|---|
| Na+ | K+ | Cl- | |||
| Control | 1 mL/kg body weight in 5 mL of 0.9% NaCl | 192.7 ± 17.4 | 4.4 ± 0.2 | 113.3 ± 3.2 | 43.6 |
| Acetazolamide | 10 mg/kg body weight in 5 mL of 0.9% NaCl | 254.5 ± 5.5b | 6.1 ± 0.2b | 155.2 ± 5.2b | 41.5 |
| C. maxima | 150 mg/kg body weight in 5 mL of 0.9% of NaCl | 203.5 ± 6.7a | 4.3 ± 0.2 | 131.0 ± 3.8a | 33.5 |
| C. maxima | 300 mg/kg body weight in 5 mL of 0.9% of NaCl | 257.2 ± 10.2b | 6.1 ± 0.1b | 148.0 ± 2.2b | 42.3 |
| Values are in mean ± SEM; ap<0.05 Vs control; bp<0.01 Vs control | |||||
These two processes are happened by the suppression of renal tubular reabsorption of electrolytes, water and low molecular weight organic compounds into the blood stream and a consequence; promote the forma tion of urine (De Stevens, 1963). This activity of C. maxima hydroalcholic extract was due to the presence of glycosides, flavonoids, polyphenols (Parial et al., 2009) and alkaloids (Chandra Kalyan et al., 2011). The activity of the extract could be dose dependent one. Further isolation and characterization needs to explore the diuretic potency of hydroalcoholic extract of C. maxima.
References
Caili FU, Shi Huan, Quanhong LI. A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods Hum Nutr. 2006; 61: 73-80.
Chandra Kalyan Reddy Y, Sandya L, Sandeep D, Salomi KR, Nagarjuna S, Reddy YP. Evaluation of diuretic activity of aqueous and ethanolic extracts of Lawsonia inermis leaves in rats. Asian J Plant Sci Res. 2011; 1: 28-33.
Chaudhri RD. Herbal drugs industry: A practical approach to industrial pharmacognosy. 1st ed. New Delhi, Eastern Publishers, 1996: 505-06.
De Stevens G. Diuretics: Chemistry and pharmacology. 1st ed. New York, Academic Press, 1963, pp 2-7, 52-58.
Divakar MC. Plant drug evaluation: A laboratory guide. 2nd ed. Coimbatore, CD Remedies, 2005, pp 42-45.
Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo ZG. Medicinal plants in therapy. Bull World Health Org. 1985; 63: 83-97.
Hancock RD, Haupt S, Viola R. Synthesis of l-ascorbic acid in phloem. Plant Biol. 2003; 3: 1-13.
Harbone JB. Phytochemical methods. 3rd ed. New Delhi, Springer (India) Pvt Ltd, 1998, pp 4-6.
Kirtikar KR, Basu BD. Indian medicinal plants. 2nd ed. India, Oriental Enterprises, 2003, pp 1606-08.
Modi DC, Patel JK, Shah BN, Nayak BS. Pharmacognostic studies of the seed of syzygium cumini Linn. Int J Pharm Sci. 2010; 1: 20-26.
Nawirska-OlszaÅ„ska A, Biesiada A, Sokół-ÅÄ™towska A, Kucharska AZ. Content of bioactive compounds and antioxidant capacity of pumpkin puree enriched with Japanese quince, cornelian cherry, strawberry and apples. Acta Sci Pol Technol Aliment. 2011; 10: 51-60
Parial S, Jain DC, Joshi SB. Diuretic activity of the extracts of Limonia Acidissima in rats. Rasayan J Chem. 2009; 2: 53-56.
Tadmor Y, Paris HS, Meir A, Schaffer AA, Lewinsohn E. Dual role of pigmentation gene B in affecting carotenoids and vitamin E content in squash Mesocarp. J Agric Food Chem. 2005; 53: 9759-63.
Thomas S, Patil DA, Patil AG, Chandra N. Pharmacognostic evaluation and physicochemical analysis of Averrhoa carambola L. fruit. J Herb Med Toxicol. 2008; 2: 51-54.
Vanamala U, Elumalai A, Eswaraiah CM, Shaik A. An updated review on diuretic plants. Int J Pharm Biol Arch. 2012; 3: 29-31.
Sahu VK, Irchhaiya R, Shashi A, Gurjar H. Phytochemical investigation and chromatographic evaluation of the ethanolic extract of whole plant extract of Dendrophthoe falcata (l.f.) Ettingsh. Int J Pharm Sci Res. 2010; 1: 39-45.
Yar MS, Ansari ZH. Synthesis and in vivo diuretic activity of biphenyl benzothiazole-2-carboxamide derivatives. Acta Pol Pharm Drug Res. 2009; 66: 387-92.
