1. INTRODUCTION:

Amritarishta is a polyherbal hydroalcoholic Ayurvedic preparation and is used as antioxidant and advised as a choice of remedy in mostly all types of fevers¹. The chief ingredient of Amritarishta is guduchi, dried stem of Tinospora cordifolia. The chemical constituents reported from stems of Tinospora cordifolia belong to different classes such as alkaloids as tinosporin^2-3, glycosides as cordifoliosides-A and cordifolioside-B^4-5, steroids as β- sitosterol⁶, sesquiterpenoid as tinocordifolin⁷ and a large amount of phenolic compounds as gallic acid, ellagic acid, catechin and epicatechin⁸. These compounds have many notable medicinal properties as antidiabetic⁹, hepatoprotective¹⁰, antioxidant¹¹, antimalarial¹², immunomodulatory¹³ and antineoplastic properties¹⁴.

However, no study has been carried out for the diuretic activity of Amritarishta in order to confirm its assumed beneficial property. Therefore, we have undertaken the present study to verify the efficacy of all the test formulations of Amritarishta as Amritarishta-T and Amritarishta-M prepared by traditional and modern methods respectively and its marketed formulation as diuretic agent in albino rats.

2. MATERIALS AND METHODS:

2.1 Preparation of Amritarishta-T:

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I¹. All the ingredients of Amritarishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Amritarishta was done by Dr. G. D. Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the Central Institute of Medicinal and Aromatic Plants, Lucknow for future reference. Identification of all the individual plant material was done as per The Ayurvedic Pharmacopoeia of India. Quantity of ingredients taken for the preparation of batch size 3.072 l of Amritarishta has been calculated according to the formula as given in The Ayurvedic Formulary of India, Part-I, 2000.

According to this method, coarsely powdered stems of guduchi (Tinospora cordifolia) with prescribed ingredients as Aegle marmelos (stem bark), Oroxylum indicum (roots), Gmelina arborea (stem bark), Stereospermum suaveolns (stem bark), Premna integrifolia (stem bark), Hedysarum gangeticum (entire plant), whole plant of Paederia foetida, entire plant of Solanum indicum, entire plant of Solanum xanthocarpum and Tribulus terrestris were placed in polished vessel of brass along with prescribed quantity of water (12.288l) and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity(3.072 l) , then the heating was stopped and it was filtered in cleaned vessel and after that jaggery was added and mixed properly. Then, prakshepa dravyas as svet jiraka, raktapuspaka, saptaparni, sunthi, marica, pippali, nagakesara, mustaka, katuka, ativisa and indravaruni in fine powdered form were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33±1°C. After 15 days completion of fermentation was confirmed by standard tests¹⁵. The fermented preparation was filtered with cotton cloth and kept in clean covered vessel for further next seven days. Then, when the fine suspended particles settled down, it is strained again and poured in amber colored glass bottles previously rinsed with ethyl alcohol, packed and properly labelled.

2.2 Preparation of Amritarishta-M:

Method of preparation of Amritarishta-M was same as followed for Amritarishta-T only in addition to jaggery, yeast was also added for inducing fermentation¹⁶.

2.3 Animals:

Adult wistar albino rats, weighing between 200-220g of either sex were acclimatized to normal environmental conditions in the animal house for one week. The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22ºC±2ºC) and humidity (55±5%) with 12:12 hour light and dark cycle. All the animals were given a standard chow diet (Hindustan Lever Limited) and water ad libitum. The guidelines of the Committee for the Purpose of Control and Supervision of Experimentals on Animals (CPCSEA) of the Government of India were followed and prior permission was granted from the Institutional Animals Ethics Committee (CPCSEA No. 07/09).

2.4. Experimental Procedure:

The method of Lipschitz et al., (1943) was employed for the assessment of diuretic activity. Twenty four hours before testing the animals were transferred to metabolic cages¹⁷.Then only water was made accessible ad libitum without food.

All the animals were randomly divided into the five groups with six animals in each group as follows:

Group I Control group received normal saline as vehicle (25 ml/kg, p.o.)

Group II Animals received furosemide (10 mg/kg, p.o.)

Group III Animals received Amritarishta-T (2 ml/kg, p.o.)

Group IV Animals received Amritarishta-M (2 ml/kg, p.o.)

Group V Animals received marketed Amritarishta (2 ml/kg, p.o.)

The second group received same volume of normal saline (25 ml) in which furosemide (10 mg/kg bw) was dissolved. The animals of Group III, IV and V received Amritarishta-T, Amritarishta-M and marketed Amritarishta at the dose of 2 ml/kg bw orally, after diluting to all of them up to 25 ml with normal saline to maintain the fluid intake same in all the cases. Immediately after dosing the rats were placed in metabolic cages and kept at room temperature of 25 ºC±0.5 ºC for 5 h. During this period, no food and water was made available to them. At the end of 5 h the animals were taken out of the cages and the total volume of urine excreted by each group was noted. Urine samples were analysed thereafter for Na⁺ and K⁺ concentration by flame photometer while chloride (Cl^-) was determined by using standard kit containing chloride reagent from span diagnostics, Surat, India.

2.5. Statistical analysis:

The results have been expressed as mean ± SEM. Statistical analysis of data among the various groups was performed by using one way analysis of variance (ANOVA) followed by the Tukey’s test using Graph Pad Prism software of Statistics. Significance value (P<0.05) was considered statistically significant.

3. RESULTS:

Diuretic effect:

Total urine output:

Both types of Amritarishta as Amritarishta-T and Amritarishta-M were prepared by traditional and modern methods respectively showed significant (P<0.001) increase in urine volume, as compared to control group. The diuresis was almost equal to that produced by furosemide (Fig.1).

Fig. 1. Effect of Amritarishta-T, M and marketed Amritarishta on urine volume

All values are expressed as mean ± SEM (n = 6); b P<0.001 as compared to control

Urinary electrolyte concentration:

Urinary sodium: All the test formulations of Amritarishta as Amritarishta-T, Amritarishta-M and its marketed formulation were found to produce significant (P<0.001) increase in natriuresis but the maximum natriuresis was produced by furosemide (Fig.2).

Urinary potassium:

Both types of Amritarishta as Amritarishta-T and Amritarishta-M have been found to produce significant (P<0.001) increase in the excretion of potassium in urine as compared to the control group. Furosemide also significantly increased the excretion of potassium. Thus, all the test formulations of Amritarishta showed significant kaliuretic effect (Fig.2).

Urinary chloride:

All the test formulations of Amritarishta as Amritarishta-T, Amritarishta-M and its marketed formulation showed significant (P<0.001) increase in the excretion of chloride in urine as compared to control. Furosemide also showed significant increase in the excretion of chloride in urine (Fig.2).

Fig. 2. Effect of Amritarishta-T, M and marketed Amritarishta on urinary electrolyte concentration

All values are expressed as mean ± SEM (n = 6); b P<0.001 as compared to control

4. DISCUSSION:

This study shows that both types of Amritarishta as Amritarishta-T and Amritarishta-M prepared by traditional and modern methods respectively and its marketed formulation produced striking increase in total urine output over a period of 5 h. All these test formulations of Amritarishta also showed significant (P<0.001) increase in the excretion of sodium, potassium and chloride in urine as compared to control group. Therefore, both types of Amritarishta as Amritarishta-T and Amritarishta-M have been shown to possess significant diuretic, natriuretic and kaliuretic effects which may be one of the basis of their therapeutic application in various ailments, such as nephritis, burning micturation etc. and different oedematous diseases. Their diuretic effects have been shown to be more or less equal to that produced by furosemide.

Preliminary phytochemical studies have confirmed the presence of phenolics, particularly hydrolysable tannins and flavonoids and other nonphenolic constituents as steroidal saponins in all the test formulations of Amritarishta as Amritarishta-T, Amritarishta-M and its marketed formulation, promoting the hypothesis that these type of polar compounds may also be responsible for the diuretic effects. It is known that this type of compounds increase renal circulation, and thus the rate of glomerular filtration which promotes increased urine formation^18-20. Thus, presence of self generated alcohol helps in the faster absorption of biologically active compounds as tannins, flavonoids and steroidal saponins which by their chemical nature are antioxidants, might contribute to the prevention of cardiac diseases as hypertension by acting as diuretics²¹

5. REFERENCES:

1) The Ayurvedic Formulary of India, Part-I. 2000, 1^st edition, The Controller of Publications, Delhi, 6.

2) Kumar S, Verma NS, Pande D and Srivastava PS. In vitro regeneration and screening of berberinein Tinospora cordifolia. Journal of Medicinal and Aromatic Plant Science 2000;22:61.

3) Biset NG and Nwaiwu J. Quaternary alkaloids of Tinospora species. Planta Medica 1983;48:275-9.

4) Maurya R, Wazir V, Tyagi A and Kapil RS. Cordifoliosides A and B, two new phenylpropene disaccharides from Tinospora cordifolia possessing immunostimulant activity. Natural Product Letter 1996;8:7-10.

5) Gangan VD, Pradhan P, Sipahimalani AT and Banerji A. Cordifoliosides A, B, C: Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 1994; 37:781-6.

6) Dixit SN and Khosa RL. Chemical investigation of Tinospora cordifolia. Indian Journal of Applied Chemistry 1971; 34:46-7.

7) Maurya R and Handa SS. Tinocordifolin, a sesquiterpene from Tinospora cirdifolia. Phytochemistry 1998; 49:1343-6.

8) Kidwai AR, Salooja KC, Sharma VN, Siddiqui S. Chemical examination of Tinospora cordifolia. Journal of Science and Indian Research 1949; 8:115-8.

9) Stanely M, Prince P and Menon VP. Antioxidant action of Tinospora cordifolia root extract in alloxan diabetic rats.Phytotherapy Research 2001; 15:213-8.

10) Mehrotra R, Katiyar CK and Gupta AP. Hepatoprotective compositions and composition for treatment of conditions related to hepatitis-B and E infection. US Patent 749296. 2000.

11) Prince PS and Menon VP. Antioxidant activity of Tinospora cordifolia roots in experimental diabetes. Journal of Ethnopharmacology 1999; 65:277-81.

12) Ikram M, Khattak SG and Gilani SN. Antipyretic studieson some indigenous Pakistani medicinal plants. Journal of Ethnopharmacology 1987; 19:185-92.

13) Manjrekar PN, Jolly CI and Narayanan S. Comparative studies of immunomodulatory activity of Tinospora cordifolia and Tinospora sinensis. Fitoterapia 2000;71:254-7.

14) Jagetia GC, Nayak V and Vidyasagar MS. Evaluation of the antineoplastic activity of guduchi (Tinospora cordifolia) in cultured HeLa cells. Cancer Letter 1998; 127:71-82.

15) Mishra S. Bhaisazya Kalpana Vigyan. Varanasi, India: Chaukambha Surbharati Prakashan; 2005.p. 253-254.

16) Alam M, Radhamani S, Ali U and Purushottam KK. Microbiological Screening of Dhataki flowers. Journal of Research in Ayurveda and Siddha 1984; 2(4):371-375.

17) Lipschitz WL, Hadidian Z, Kerpcsar A. Bioassay of Diuretics. Journal of Pharmacology and Experimental Therapeutics 1943; 79: 97-110.

18) Afzal M, Khan NA, Ghufran A, Iqbal A, Inamuddin M. Diuretic and nephroprotective effect of Jawarish Zarooni Sada- a polyherbal Unani formulation. Journal of Ethnopharmacology 2004;91:219-223.

19) Loew D, Heimsoth V, Erwin K, Schilcher H. 1991. Diureticos: Quimica, Farmacologiay Therapeutica incluida Fitoterapia, Barcelona, Salvat Editores S.A.:270.

20) Das PK, Goswami S, Chinniah A. Woodfordia fruticosa: Traditional uses and recent findings. Journal of Ethnopharmacology 2007; 110:189-199.

21) Hollman PCH, Katan MB. Dietary Flavonoids: Intake, Health effects and bioavailability. Food and Chemical Toxicology 1999; 37:937-942

Received on 26.11.2013 Modified on 13.01.2014

Res. J. Pharmacology & P’dynamics. 6(2): April- June 2014; Page 71-74