|Year : 2011 | Volume
| Issue : 6 | Page : 714-717
Natriuretic and saluretic effects of Hemidesmus indicus R. Br. root extracts in rats
Navneet B Gadge, Sunil S Jalalpure
Department of Pharmacognosy and Phytochemistry, KLE University's College of Pharmacy, JNMC Campus, Nehru Nagar, Belgaum- 590 010, Karnataka, India
|Date of Submission||17-Sep-2010|
|Date of Decision||06-Jun-2011|
|Date of Acceptance||31-Aug-2011|
|Date of Web Publication||14-Nov-2011|
Navneet B Gadge
Department of Pharmacognosy and Phytochemistry, KLE University's College of Pharmacy, JNMC Campus, Nehru Nagar, Belgaum- 590 010, Karnataka
Source of Support: ICMR, New Delhi, Conflict of Interest: None
Objective : The present study was aimed to investigate the diuretic effects of aqueous (AqE) and ethanolic (EtE) crude extracts of Hemidesmus indicus R. Br. roots (family - Asclepiadaceae) using acute model in rats.
Materials and Methods : A single individual dose of AqE and EtE of H. indicus root (200 mg/kg and 400 mg/kg, p.o., each), frusemide and hydrochlorothiazide, (25 mg/kg, p.o., each) as reference diuretic drugs were administered orally to dehydrated rats. Control group rats were fed with normal saline (25 ml/kg, p.o.). All rats were caged in metabolic cages in a pairs and their urine output was monitored at 5 and 24 h intervals.
Results : Both extracts significantly increased the urine output in higher doses. Although, the onset of this diuretic action was gradual (within 5 h), it lasted throughout the studied period (up to 24 h). Further, the intensity of diuresis induced by AqE (400 mg/kg) in 5 h was almost similar to that of frusemide and hydrochlothiazide. AqE of H. indicus root also caused marked increase in urinary Na + and K + levels. However, the routine urinalysis showed non-significant alterations in pH and specific gravity by either dose of crude extracts of H. indicus roots.
Conclusions : These effects demonstrate possible diuretic actions of H. indicus root extracts and support its folklore use in various urinary ailments. Further study need to be done to characterize active phytoconstituents.
Keywords: Diuretic, frusemide, Hemidesmus indicus, hydrochlorthiazide
|How to cite this article:|
Gadge NB, Jalalpure SS. Natriuretic and saluretic effects of Hemidesmus indicus R. Br. root extracts in rats. Indian J Pharmacol 2011;43:714-7
| » Introduction|| |
Hemidesmus indicus R. Br. (Family - Asclepiadaceae), commonly known as 'Indian sarsaparilla', is a well known drug in Ayurveda. Roots of H. indicus are considered to be tonic and diuretic and used traditionally by Indian tribes for a wide range of ailments, including nutritional disorders, skin diseases, gravel, and other urinary problems. Various classical formulae include an infusion or decoction of root or powdered root mixed with cow's milk that give benefits in cases of scanty and highly colored urine and in those of gravel and strangury. , Isolation of nine pregnane glycosides viz. desinine,  indicine, hemidine,  indicusin,  hemidescine, emidine,  medidesmine, hemisine, and emicine  from H. indicus have been reported along with certain important triterpenoids (including alpha-amyrin, beta-amyrin, lupeol, lupanone, hexadecanoic acid) and 3-hydroxy-4-methoxy benzaldehyde. 
Pharmacologically, it is shown to possess antinociceptive, antioxidant, renoprotective, and hepatoprotective activity. ,,, So far the diuretic potential of this plant has not been established scientifically. Hence, the present study was undertaken to assess the acute diuretic activity of H. indicus roots in rats.
| » Materials and Methods|| |
Collection of Plant Material and Preparation of Extracts
Roots of H. indicus were collected during September 2008, from Jamboti forest in Belgaum district, Karnataka, India, and authenticated by Dr. P. G. Diwakar at Botanical Survey of India (BSI), Pune, Maharashtra. A voucher specimen of the plant (NBGHIP-1) was deposited in the BSI herbarium.
For the preparation of ethanolic extract (EtE), the shade dried powdered roots (500 gm) were extracted with 95% v/v ethanol using soxhlet assembly. Using another lot of shade dried powdered roots (500 gm), an aqueous extract (AqE) was prepared using maceration method for seven days at room temperature.
Further, each extract was concentrated using rotary flash evaporator under vacuum to semi-solid mass. Finally, the residue was dried in desiccator over sodium sulphate (Yield: EtE = 14.23% w/w, AqE = 12.04% w/w). For oral administration in rats, a suspension of AqE and EtE was prepared using 0.5% w/v aqueous sodium carboxymethyl cellulose as vehicle.
Healthy inbred male Wistar rats weighing between 150 to 200 g were procured from Shri Venkateshwara Enterprises, Bangalore, Karnataka. They were acclimatized to standard laboratory conditions and maintained on 12:12 h, light:dark cycle. They were fed regular rat chow and drinking water ad libitum. The Ethics Committee of KLE University's College of Pharmacy, Belgaum, Karnataka, approved the protocol of the study (CPCSEA Registration no. is 627/02/a/CPCSEA).
Acute Oral Toxicity Study
The acute oral toxicity study was carried out as per the guidelines No. 423 set by organization for economic co-operation and development (OECD).  Based on the cut-off value of the median lethal dose (LD 50 ), the therapeutically effective dose was derived.
Assessment of Diuretic Activity
The method of Lipschitz et al. (1943) with some modifications was used to study the diuretic activity in male Wistar rats.  Rats, fasted and deprived of water for 18 h prior to the experiment were divided in seven groups containing eight rats in each. Group 1, serving as control, received vehicle (25 ml/kg). Group 2 and 3 received frusemide (25 mg/kg) and HTZ (25 mg/kg) in vehicle as reference diuretic drugs, respectively.  Groups 4 and 5 received the AqE (200 and 400 mg/kg), respectively and group 6 and 7 received the EtE (200 and 400 mg/kg), respectively. All groups received a single individual dose by oral route using gastric intubation method.
Collection and Analysis of Urine
Immediately after dosing, a pair of two rats per cage was kept in metabolic cages and urine was collected at 5 h and 24 h intervals. Routine urinalysis including determination of pH and specific gravity along with presence of occult blood, bilirubin, urobilinogen, ketone bodies, proteins, nitrite, glucose, and leucocytes in urine was carried out using urocolor test strips (Standard Diagnostics Inc., South Korea) for urine samples of control and extract treated rats. Urine volume (ml/100 g) and Na + , K + and Cl - concentrations (mEq/l/100 g) in the urine were determined  and various indices for diuretic action were calculated. ,,
Diuretic index = urine volume of test group/urine volume of control group
Saluretic index = urinary excretion of electrolyte of test group/urinary excretion of electrolyte of control group
Natriuretic index = urinary excretion of Na + /urinary excretion of K +
Ion quotient = urinary excretion of Cl - /sum of urinary excretion of Na + and K +
Results were expressed as mean ± SEM. Significance of difference between control and treated group was determined by unpaired Student's t-test. Level of significance was kept at P < 0.05.
| » Results|| |
Orally administered to rats with starting dose of 2000 mg/kg, AqE and EtE of H. indicus increased urination and defecation. No mortality was observed with starting dose. However, two rats among each group died with the confirmatory dose of both extracts. Further, treatment with the lower dose (300 mg/kg, p.o.) of AqE and EtE was found to be safer and the mortality rate was reduced to zero. Hence, therapeutic doses were calculated from 1/10 th and 1/5 th of the LD 50 cut-off dose (2000 mg/kg) for both extracts.
[Table 1] shows the effect of oral administration of AqE and EtE in rats. The results clearly indicate that no abrupt change in pH and specific gravity was observed in any of the extract treated groups. Also, the urinary excretion of bilirubin, urobilinogen, leucocytes, ketone bodies, and nitrite were found to be below detectable limits among all experimental groups (data not given). Except with EtE (400 mg/kg) showing traces of proteins in urine, other groups were devoid of preoteinuria and glucosuria.
|Table 1: Effect of Hemidesmus indicus root extracts on miscellaneous urinary parameters in control and experimental rats|
Click here to view
Among the reference diuretic drugs, frusemide and HTZ induced excretion values of water of more than 100%, when compared to the untreated control group [Table 2]. Both crude extracts significantly increased the urine output in a dose dependent manner. A gradual onset of diuretic action was observed within first 5 h comparable to all three reference diuretic drugs. This effect lasted up to 24 h showing significantly (P < 0.001) increased urine excretion volume [Figure 1]. The intensity of diuresis induced by AqE (400 mg/kg) in 5 h was almost similar to that of frusemide and HTZ [Table 2]. In comparison with EtE, the AqE showed higher diuretic index.
|Table 2: Effect of Hemidesmus indicus root extracts on urine excretion volume|
Click here to view
|Figure 1: Cumulative urine excretion (ml/100 g) in control, standard and experimental groups; Values are expressed as mean ± SEM at 5 and 24 h intervals; Non-signifi cant P > 0.05, *P < 0.05, **P < 0.01,|
***P < 0.001; Two way ANOVA followed by Bonferroni post test
Click here to view
An increase in the urinary excretion of electrolytes (Na + , K + and Cl - ) between 20 to 90% exhibited by the reference diuretic drugs over 24 h period was found to be significant (P < 0.01) in comparison with the control group [Table 3]. Both extracts of H. indicus root showed significant increase in urinary Na + and Cl - levels and a reduction in the osmolarity of urine. Except with AqE (400 mg/kg) showing a significant (P < 0.05) increase in urinary excretion of K + , the kaliuretic changes with other extracts were non-significant. An increase in Na + excretion shown by AqE over a period of 24 h was significant (P < 0.01) compared to control group. Although EtE administration resulted in moderate diuresis, the rise in urinary electrolyte excretion was less than 30%; whereas, AqE treatment showed nearly 30 to 70% increase in urinary electrolyte excretion when compared to control group [Table 3]. Moreover, the higher dose (400 mg/kg) of AqE showed significant saluretic activity.
|Table 3: Effect of Hemidesmus indicus root extracts on urinary excretion of electrolytes|
Click here to view
| » Discussion|| |
Diuresis occurs by two phenomena including net increase in urine volume (water excretion) and elevated excretion of electrolytes (solutes) in the urine.  These processes result from suppression of renal tubular reabsorption of water and electrolytes into the blood stream. The thiazide diuretics inhibit Na + /Cl - symporter (co-transporter system) in the distal convoluted tubule by competing for the Cl - binding site and thereby increasing the excretion of Na + and Cl - , while the loop diuretic reference drug, frusemide, increases the urine output and urinary excretion of Na + by inhibiting Na + /K + /Cl - symporter in the thick ascending limb of loop of Henle. 
In the present study, 25 mg/kg dose of frusemide and HTZ, each, showed significant diuresis in rats over a period of 24 h. In comparison, similar increase in urine excretion was found with the AqE and EtE of H. indicus, when administered orally in dehydrated rats [Table 2]. While, the different results obtained on electrolytic excretion by both extracts of H. indicus suggest a difference in their comparative diuretic profile. AqE and EtE show a gradual rise in excretion of electrolytes (Na + , K + and Cl - ) in a dose-dependent manner. Particularly, the EtE show unsubstantial rise in urinary K + levels. Further, there was no alkalization of urine. Collectively, these observations suggest that the EtE act as potassium-sparing diuretics in agreement with its natriuretic index. ,,
Although the thiazide diuretics, like HTZ, increase only the urinary K + level and alter the urinary Na + /K + ratio, , the administration of AqE in this study caused increase in urinary K + levels along with urinary Na + and Cl - levels without significant alteration in the Na + /K + ratio. Such activity of AqE reflects that is unlikely to predict the mechanismof AqE as thiazide or loop diuretics. Moreover, evidences suggest that AqE in high dose (400 mg/kg) act by kaliuretic action in conjunction with its saluretic activity. Further, the intensity of aquaresis and accompanied marked increase in urinary Na + and K + levels by the AqE of H. indicus roots were similar to that of furosemide and HTZ. Also, changes in urine pH were non-significant. These features strongly suggest that the AqE act more as a loop diuretic than as thiazide diuretic, mainly due to its higher natriuretic, kaliuretic, and saluretic actions. Loop diuretics mainly inhibit the Na + /K + /Cl - symporter in the thick ascending loop of Henle; thereby cause to increase natriuresis and kaliuresis. ,, Nevertheless, moderate acidification of urine is also seen with these diuretics. ,, Besides another important index, viz. the ion quotient in between 0.8 to 1.0 as exhibited by the experimental groups suggest their unequivocal association with carbonic anhydrase inhibition.  The onset of the diuretic activity of the AqE and EtE was gradual till first 5 h after dose administration which is in conformity with clinically used synthetic loop diuretics. , Interestingly, in spite of the heavy loss of urinary Na + and K + , there was a significant reduction in the osmolarity of urine in AqE and EtE treated rats. Considering the fact that inhibition of ADH causes polyurea with low osmolarity, , it is possible that the AqE and EtE induced diuresis in the present study may also be due to the impaired basal secretion of ADH and/or diminished sensitivity of uriniferous tubules to the action of ADH.
High ceiling loop diuretics are clinically used in patients with salt and water overload due to host of conditions. The observed mode of action of AqE and EtE in the present study indicate that traditional practitioners may find H. indicus roots being useful as a non toxic natural therapeutic agent in the treatment of conditions such as pulmonary edema, cardiac edema, hypertension etc. , Besides The only limitation of increased risk of hypokalaemia, as with other therapeutically used loop diuretics, the diuretic actions of crude extracts of H. indicus roots conclude that the plant as an appealing alternative to presently available diuretic drugs. Though the onset of the diuretic action of the AqE and EtE was fairly gradual, it had a substantially prolonged duration of action. Thus, it is probable that it would curtail the frequency of administration of a diuretic drug.
The findings of this study support the folklore use of Hemidesmus indicus roots for their diuretic actions. The plant extracts at the dose studied do not seem to have renal toxicity in rats. Although, the phytochemistry of active components remain unidentified based on the pattern of excretion of water and electrolytes, it appears that that there are at least two types of active principles present in these extracts, one having a frusemide-like activity and the other a HTZ-like activity. However, further systematic phytochemical studies are necessary to elucidate the probable structure activity relationship of biomolecules.
| » References|| |
|1.||Nadkarni AK. Indian materia medica. Vol.1. Bombay: Popular Book Depot; 1976. p. 619-22. |
|2.||Kirthikar KR, Basu BD. Indian medicinal plants. Vol. 3. Dehradun: International Book Distributors; 1999. p. 1596-8. |
|3.||Oberai K, Khare MP, Khare A. A pregnane ester diglycoside from Hemidesmus indicus. Phytochemistry 1985;24:2395-7. |
|4.||Prakash K, Sethi A, Deepak D, Khare A, Khare MP. Two pregnane glycosides from Hemidesmus indicus. Phytochemistry 1991;30:297-9. |
|5.||Deepak D, Srivastava S, Khare A. Indicusin - A pregnane diester triglycoside from Hemidesmus indicus R. Br Nat Prod Lett 1995;6:81-6. |
|6.||Chandra R, Deepak D, Khare A. Pregnane glycosides from Hemidesmus indicus. Phytochemistry 1994;35:1545-8. |
|7.||Deepak D, Srivastava S, Khare A. Pregnane glycosides from Hemidesmus indicus. Phytochemistry 1997;44:145-51. |
|8.||Gupta MM, Verma RK, Misra LN. Terpenoids from Hemidesmus indicus. Phytochemistry 1992;31:4036-7. |
|9.||Prabakan M, Anandan R, Devaki T. Protective effect of Hemidesmus indicus against rifampicin and isoniazide-induced hepatotoxicity in rats. Fitoterapia 2000;71:55-9. |
|10.||Ravishankara MN, Shrivastava N, Padh H, Rajani M. Evaluation of antioxidant properties of root bark of Hemidesmus indicus R. Br. (Anantmul). Phytomedicine 2002;9:153-60. |
|11.||Kotnis MS, Patel P, Menon SN, Sane RT. Renoprotective effect of Hemidesmus indicus, a herbal drug used in gentamicin-induced renal toxicity. Nephrology (Carlton) 2004;9:142-52. |
|12.||Verma PR, Joharapurkar AA, Chatpalliwar VA, Asnani AJ. Antinociceptive activity of alcoholic extract of Hemidesmus indicus R. Br. in mice. J Ethnopharmacol 2005;102:298-301. |
|13.||Anonymous. OECD series on testing and assessment, Number 24. Guidance document on acute oral toxicity testing. ENV/JM/MONO (2001) 4. Paris: Environment directorate, OECD; 2001. |
|14.||Lipschitz WL, Haddian Z, Kerpscar A. Bioassay of diuretics. J Pharmacol Exp Ther 1943;79:97-110. |
|15.||Swain SR, Sinha BN, Murthy PN. Antiinflammatory, diuretic and antimicrobial activities of Rungia pectinata Linn. and Rungia repens Nees. Indian J Pharm Sci 2008;70:679-83. |
|16.||Martin-Herrera D, Abdala S, Benjume D, Gutierrez-Luis J. Diuretic activity of some Withania aristata Ait. fractions. J Ethnopharmacol 2008;117:496-9. |
|17.||Mukherjee PK. Evaluation of diuretic agents. Quality control of herbal drugs. New Delhi: Business Horizons; 2002. |
|18.||Jackson EK. Drugs affecting renal and cardiovascular function. In: Hardman JC, Gilman AG, Limbird LE, editors. Goodman and Gilman's the pharmacological basis of therapeutics. 9 th ed. New York: Pergamon Press; 1996. p. 685-713. |
|19.||Rang HP, Dale MM, Ritter JM. Pharmacology. London: Churchill Livingstone; 1995. p. 367-84. |
|20.||British National Formulary. London: British Medical Association and Royal Pharmaceutical Society of Great Britain; 2000. p. 63-9. |
|21.||Kreydiyyeh SI, Usta J. Diuretic effect and mechanism of action of parsley. J Ethnopharmacol 2002;79:353-7. |
|22.||Osorio FV, Teitelbaum I. Mechanisms of defective hydroosmotic response in chronic renal failure. J Nephrol 1997;10:232-7. |
|23.||Mayne PD. Clinical chemistry. London: Edward Arnold; 1994. p. 2-24. |
[Table 1], [Table 2], [Table 3]