IJSPER , Vol.
1 (11), October 2012 (8-13)
(ISSN: 2277 2561)
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DIURETIC ACTIVITY OF THE AQUEOUS EXTRACT OF NARDOSTACHYS JATAMANSI DC IN NORMAL RATS.
Velpandian Venkatachala Pathy 1*, Banumathi Vellaian 1, Mohammed Musthafa2, Anbu Natarajan 2
1
Post Graduate department of Gunapadam (Pharmacology), Govt.Siddha Medical College, Chennai 106. 2 Post Graduate department of Maruthuvam (Medicine), Govt.Siddha Medical College, Chennai 106.
ABSTRACT
In the Indian System of medicine, the rhizome of Nardostachys jatamansi (NJ) is more commonly used as diuretics. Though studies have been conducted on different activities of NJ, the diuretic activity of this plant has not been investigated in scientifically controlled studies. Therefore the present study was planned to evaluate the diuretic potential and effect on urinary electrolytes of aqueous NJ rhizome extract in normal albino rats. Different concentrations of this plant extract (100 mg, 150 mg and 200 mg/kg of body weight) and the standard drug Furosemide (10 mg/kg) were administrated orally to hydrated male Wistar rats and their urine output was measured at several interval of time after a single dose administration. After single dose of the extract of NJ was found to possess significant dose dependent diuretic activity. The parameters measured for diuretic activity were total urine volume, sodium, potassium and chloride content. Urine output was significantly increased at all time points. The NJ extract did not appear to have renal toxicity or any other adverse effects during the study period. In conclusion, aqueous extracts of Nardostachys jatamansi has strong diuretic action confirming their traditional use. Key words: Nardostachys jatamansi, diuretic activity, saluretic, Jadamanjil, electrolytes, furosemide. INTRODUCTION Diuretics are drugs which reduce the oedema due to salt and water retention in disorders of heart, kidney, liver or lungs. These agents are also useful in reducing the symptoms of volume overload, including orthopnoea and paroxysmal nocturnal available online on www.ijsper.com dyspnoea. Diuretics decrease plasma volume and, subsequently, decrease venous return to the heart. This decreases the cardiac workload and the oxygen demand. Diuretics may also decrease afterload by reducing plasma volume, thus decreasing blood pressure [1] . So the diuretic drugs are used
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IJSPER , Vol.1 (11), October 2012 (8-13)
(ISSN: 2277 2561)
MATERIALS AND METHODS Collection, Identification and Preparation of Plant extract (NJE) The raw drug of Nardostachys jatamansi was obtained from country shop at Chennai, Tamilnadu, authenticated at Department of Gunapadam (Pharmacology), Govt.Siddha Medical College, Arumbakkam, Chennai and a voucher specimen has been preserved in the department for future reference. After collection and identification, the rhizomes of Nardostachys jatamansi were cleaned well and dried for a week in the shade. The purified rhizomes were made into a fine powder form-Choornam and it was filtered by white cloth. To 20 g of each dried plant powder form, 500 ml water was added and contents of flask were mixed thoroughly by gentle shaking. Flasks were kept for four days with frequent shaking. After the completion of maceration process the filtrate was obtained and allowed to evaporate water to get the dried extract (evaporation by keeping flasks in electric mantle at 800 C) [9]. The residual extract was dissolved in water and was used in the preclinical studies. The freshly prepared solution of NJE was used in each experiment. PHARMACOLOGICAL ACTIVITY Animals Healthy adult albino rats of either sex weighing about 150-180 g were selected for the screening of diuretic activity in vivo. All animals were housed in standard metallic cages (6 rats per cage). The animals were acclimatized to standard laboratory conditions (temperature: 253C) and maintained on 12-h light: 12-h dark cycle. They were provided with regular rat chow and drinking water ad libitum. The study was conducted in accordance with CPCSEA (Committee for the Purpose of control and supervision of Experiment on Animals) guidelines and the study was approved by the IAEC (Institutional Animal Ethical Committee).
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frequently in combination with other hypotensive drugs in the treatment of hypertension. Due to the demand or lack of attention of natural diuretic drugs from plants, most of the present drugs are synthetic in nature. Although extensive work is being carried out, still, there is a need for more effective and less toxic diuretics from nature [2]. There are very few studies attempted to find the mechanism involved in the diuretic action of plant extracts. It is therefore, planned to study the effects of Nardostachys jatamansi plant extract for its diuretic activity and to explore the involvement of particular mechanism in this physiological activity. Nardostachys jatamansi DC belongs to the family Valerianceae. It is commonly known as Indian spikenard or musk root and it is a native of Himalayan region, available in Deccan plateau also. Nardostachys jatamansi is used traditionally in the treatment of hypertension, pulmonary congestion, peripheral oedema, excitement, epilepsy, insomnia and depressive illness [3] . More than 25 active principles have been isolated from the rhizome part of this plant which includes jatamansic acid, jatamansone and nardostachone [4-7]. This plant has demonstrated several pharmacological activities including sedative, anti spasmodic, hepato protective, cardio protective and hypolipidemic and antifungal [8] . Though studies have been conducted on different activities of Nardostachys jatamansi, no information is available on diuretic activity of the plant. With this background the diuretic activity of Nardostachys jatamansi was carried out to find its possible effect on urinary excretion including urine sodium, potassium and chlorine excretion.
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IJSPER , Vol.1 (11), October 2012 (8-13)
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potassium concentrations were measured by flame photometry [14-15] and Chlorine concentration was estimated by titration with silver nitrate solution (N/50) using 1 drop of 5% potassium chromate solution as indicator[16] . Furosemide sodium salt was given by stomach tube. Optimal dose activity relation was found to be 20mg/kg of furosemide per kg body weight in series of supportive experiments. STATISTICAL ANALYSIS The result were expressed as a mean+ SEM the difference were compared using One Way Analysis of Variance (ANOVA) and subsequently followed by Bonferronis multiple comparison test. P values less than 0.05 were considered statistically significant. Table No.1 - Effect of aqueous extract of Nardostachys jatamansi on urine output in rats Group (n = 6) Urine pH volume in ml 2.580.06 6.18 *** 6.530.17 7.23 Diuretic index (T / C) -2.53
ACUTE TOXICITY The Acute toxicity study of NJE was evaluated in rats as per the OECD guide lines 423 [10]. As per the guideline this test was carried out based on stepwise procedure with the use of the minimum number of animal per step. Three animals were used for each step. The dose level of 5, 50, 300 and 2000 mg/kg body weight was administered stepwise. Observations were made and recorded systematically and continuously observed as per the guideline after substance administration. The number of survivors was noted after 24 hours and these animals were then maintained for a further period of 14 days and observations were made daily [11] . One tenth and one twentieth dose was considered for diuretic activity. DIURETIC STUDY Diuretic study was carried out by Lipchitz method [12]. In this method 24 male Wistar albino rats weighing about 150 to 180 gm were selected. The animals were grouped into four of six animals each. They were fasted and deprived of water for eighteen hours prior to the experiment. Group I received normal saline (25ml/kg) and served as control. Group II received furosemide 20 mg/kg as standard. Group III and IV received test drug NJE at the doses of 100 mg/kg and 200 mg/kg respectively [13]. The extract of different doses was formulated as a suspension in normal saline and was administered orally using intra gastric tube. After administration animals were placed in metabolic cages (6 in each cage) kept at room temperature of 25 0.5C. During the period food or water was not provided to the animals. Extreme care was taken to avoid the contamination of urine with faecal matter. The urine was collected in measuring cylinder up to 5hrs after dosing. The total volume of urine collected was measured for both control and treated groups. The parameters taken for individual rat were body weight (before and after test period), total urine volume, urine sodium and available online on www.ijsper.com
CONTROL
FUROSEMIDE NJE 100 3.950.08** 6.97 1.53 NJE 200 6.060.09*** 7.19 2.36 Values are expressed as mean += S.E.M. (n=6). **p<0.01 and ***p<0.001 compared to normal (Bonferronis multiple comparison test).
Figure 1: the effect of NJE of 100 and 200 mg/kg orally on urine output (ml) results expressed as Mean += SEM, (n=6). ** p<0.01 and ***p<0.001 statistically significant when
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IJSPER , Vol.1 (11), October 2012 (8-13)
(ISSN: 2277 2561)
the evaluation carried out are listed in Table 1 and Table 2. Table 1 shows that the urine volume collected for 24 hours and other diuretic parameters such as pH and diuretic index for control group, furosemide and trial drug NJE treated orally at dose levels of 100 and 200 mg/kg. Table 2 shows the parameters related to electrolyte excretion (Na+, K+ and Cl concentrations in mEq/L), saluretic index and Na+/K+ ratio of the urine of the animals. Effect on urine volume The aqueous extract of Nardostachys jatamansi induced significant increase in urine volume with two dose levels as compared to control group (P < 0.05). Two dose levels of NJE (100 mg/kg and 200 mg/kg) were selected for study and the urine volume were found to be 3.950.08 and 6.060.09 respectively. Furosemide (25 mg/kg) treated group was found to be 6.530.17. The aqueous extract of Nardostachys jatamansi showed a dose-dependent increase in urine excretion. With respect to the aqueous extract, the maximum increase in urinary excretion was produced at 200mg/kg of NJE with a value of 6.06 ml compared with the furosemide (20 mg/kg) showed an increase of 6.53 ml in urine volume which was very much closer to the standard drug furosemide. The diuretic index was almost equal to standard drug as shown in Table 1. At dose of 200 mg/kg, NJE showed high urinary pH which was closer to standard group as shown in Table 1. From the above results it can be observed that the aqueous extract of Nardostachys jatamansi has shown significant diuretic activity by increasing urine output and was found to be dose dependent compared to control group, i.e., among the two doses studied, NJE 200mg/kg produces more effect than the lower dose. Effect on urinary electrolyte excretion The effect of standard drug furosemide and different doses of NJE on electrolyte (Na+, K+
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compared with control by ANOVA followed by Bonferronis multiple comparison test. The effect of NJE of 100 and 200 mg/kg orally on urine electrolyte excretion results expressed as Mean += SEM, (n=6). ** p<0.01 and ***p<0.001 statistically significant when compared with control by ANOVA followed by Bonferronis multiple comparison test.
Urine Electrolyte excretion
200 150
mEq/l
100 50 0
Groups
Figure 2: the effect of NJE of 100 and 200 mg/kg orally on urine electrolyte excretion (mEq/l) results expressed as Mean += SEM, (n=6). ** p<0.01 and *p<0.001 statistically significant when compared with control by ANOVA followed by Bonferronis multiple comparison test.
RESULTS AND DISCUSSION In acute toxicity study, no mortality or toxicity was observed during the entire period of study and considered to be safe orally up to the dose level of 2000 mg/kg body weight. No major behavioural changes were noted during the study. Since the plant Nardostachys jatamansi has traditional use as a diuretic, the effect of aqueous extract of the plant, standard drug furosemide and control group on urination and other parameters related to diuretic assay were investigated in albino rats and the results of
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IJSPER , Vol.1 (11), October 2012 (8-13)
(ISSN: 2277 2561)
reduction of anti diuretic hormone secretion or inhibition of carbonic anhydrase enzyme, or by producing inhibition of tubular reabsorption of water and anions. The exact mechanism of diuretic activity exhibited by the extract can only be established after more investigations of the extract and screening of diuretic activity of isolated active principles. ACKNOWLEDGEMENT The authors are grateful to the Principal and HOD, Department of Gunapadam (Pharmacology) of Government Siddha Medical College, Chennai for providing all the support and guidance. REFERENCES 1)Finkel R, Clark MA, Cubeddu LX. Lippincotts Illustrated Reviews: th Pharmacology 4 Edn, Lippincott Williams & Wilkins, Florida, 2009, 190. 2)Maryam Mirza. Diuretic activity of certain plants used in traditional system of medicine, Department of physiology, University of Karachi, Pakistan, 2004, 01-02. 3)Nadkarni AK, Indian Materia Medica, Edn 3, Vol. 1, Popular Prakashan, Mumbai, 1982, 840-842. 4)Kirthikar KR, Basu BD. In: Indian Medicinal Plants. Mahendra Pal Singh BS, editor. Vol. 2. Dehra Dun: 1993, 1307. 5)Vogel HG, Editors. Drug Discovery and Evaluation - Pharmacological Assays. 2nd Edn. Berlin, New York: Springer Verlag; 2002, 759-867. 6)Chatterjee B, Basak U, Datta J, Banerji A, Neuman, T. Prange. Studies on the Chemical Constituents of Nardostachys jatamansi DC (Valerianaceae). Cheminform 2005, 36:17.
and Cl) excretion in 24 hours urine is shown in Table 2. All doses of NJE were enhanced the excretion of the electrolytes which was found to be Na+ (115.501.38 and 133.331.68), K+ (69.501.38 and 82.330.49) and Cl (125.161.92 and 161.331.92) respectively. The dose of 100 mg/kg aqueous extract produced a moderate increase in Na+, K+ and Cl excretion, compared with the control group (P < 0.01). The dose of 200 mg/kg aqueous extracts showed significant increase (p<0.001) in excretion of sodium, potassium and chloride ions in the urine to an extent similar to that of furosemide (Na+ = 144.161.53, K+= 88.331.54 and Cl = 176.670.89). The Na+/ K+ excretion ratio was uniform (1.44 to 1.66) in all the groups studied. It should also be noted that maximum excretion was observed in animals receiving the highest dose of NJE (200 mg/kg) These results also demonstrated a dose-dependent relation for the excretion of Na+ and K+ and Cl when compared to control group. After oral administration of two different doses of NJE, standard drug furosemide and control groups, the saluretic index were noted which was found to be dose dependent effect and the saluretic effect of NJE was nearer to the standard group. CONCLUSION From this study it can be suggested that the aqueous extract of Nardostachys jatamansi is an effective and significant hypernatraemic, hyperchloraemic and hyperkalaemic diuretic with dose dependent effect with values which were very close to the standard drug furosemide, which supports the claim about the Nardostachys jatamansi being used as a diuretic in Siddha system of medicine. Though the extract appeared to cause good diuresis at different dose levels, the actual mode of action, which may be due to its effects either on loop permeability or by stimulation of regional blood flow or initial vasodilatation, or available online on www.ijsper.com
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IJSPER , Vol.1 (11), October 2012 (8-13)
(ISSN: 2277 2561)
13)Shannon Reagan-Shaw, Minakshi Nihal and Nihal Ahmad. Dose translation from animal to human studies revisited. The FASEB Journal; 2007, (22): 659-661. 14)Jeffery GH, Bassett J, Mendham J, Denny RC, Vogel's Textbook of quantitative Chemical Analysis. 5th Edition, England: Addison Wesley Longman Ltd; 1989, 801. 15)Mukherjee PK. Quality control of herbal drugs. New Delhi: Business Horizons; 2002. Evaluation of diuretic agents. 16)Beckette AH and Stenlake JB, Practical Pharmaceutical Chemistry, Part I, 1st Edition, CBS Publishers and Distributors, New Delhi, 1997, 197.
7)Hoerster H, Ruecker G, Tautges J. Valeranone content in the roots of Nardostachys jatamansi and Valeriana officinalis. Phytochem 1977, 1:1070-1071. 8)Girgune JB, Jain NK, Garg BD. Antifungal activity of some essential oils, 2. Indian Drugs. 1978, 16:2246. 9)Copper and Gunns. Tutorial Pharmacy, Edited by J.Carter 6th Edition, 2005, 257259. 10)OECD Test Guideline 423, OECD Guideline for Testing of Chemicals. Available: [http://www.oecd.org/document/html], (2001). 11)Ghosh MN, Fundamentals of Experimental Pharmacology, 2nd Edition, Scientific Book Agency, Kolkata, 1984, 14458. 12)Lipschitz WL, Hadidian Z, Kerpcar A. Bioassay of Diuretics. J.Pharmacol Exp Ther 1943, 79: 97-110.
Date of Submission: 20/08/12 Date of Acceptance: 26/09/12 Conflict of Interest: Nil Source of Support: Nil
Table No.2 - Effect of aqueous extract of Nardostachys jatamansi on electrolyte excretion in rats
+ +
Group (n = 6) Na
+
Electrolyte excretion mEq/l K
+
Saluretic index
+ +
Na /K ratio
Cl
Na
Cl
CONTROL STANDARD NJE 100 NJE 200
84.161.26 144.161.53
**
58.161.42 ** 88.331.54 69.501.38 ** 82.330.49
*
96.60.7 ** 176.670.89 125.161.92 ** 161.331.92
**
**
1.71 1.37 1.58
1.51 1.19 1.41
1.82 1.29 1.67
1.44 1.63 1.66 1.61
115.501.38 ** 133.331.68
**
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