Fitoterapia 79 (2008) 544 – 547 www.elsevier.com/locate/fitote
Preliminary study on crystal dissolution activity of Rotula aquatica, Commiphora wightii and Boerhaavia diffusa extracts Ashwinikumar A. Raut ⁎, Sudha Sunder, Subrata Sarkar, Nancy S. Pandita, Ashok D.B. Vaidya Bharatiya Vidya Bhavan's Swami Prakashananda Ayurveda Research Centre, 13th North–South Road, Juhu, Mumbai-400 049, India Received 11 January 2007; accepted 3 June 2008 Available online 1 July 2008
Abstract Several Ayurvedic plants are known to have activity against diverse urinary crystals. The traditional knowledge of Ayurveda, collective clinical experience in arthritis and the earlier experimental studies on urinary crystals led to the selection of three plants, viz. Rotula aquatica, Commiphora wightii Bhandari syn. C.mukul. and Boerhaavia diffusa for screening anticrystal activity against basic calcium phosphate (BCP), calcium pyrophosphate (CPPD) and monosodium urate monohydrate (MSUM). The effects of each plant were assayed on microcrystals in 24-well microplates in vitro. Our results show that the aqueous extracts of only R. aquatica and C. wightii have shown crystal dissolving activity against MSUM. © 2008 Elsevier B.V. All rights reserved. Keywords: Boerhaavia diffusa; Commiphora wightii; Rotula aquatica; Crystal-arthropathy; Anticrystal activity
1. Introduction Crystals of monosodium urate monohydrate (MSUM), calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP), are known to induce arthropathy. There is emerging evidence that these crystals are responsible for propagation of synovitis, progress of arthritis, cartilage damage and joint destruction [1]. Gout is the major prototype of crystal-related arthritis characterized by hyperuricaemia, arthritis and deposition of MSUM in joints. Presently, colchicine is the only specific drug available for acute gouty arthritis and other crystalrelated articular inflammations [2]. However, the drug is not extensively prescribed due to adverse side-effects. Also, at times, colchicine alone is not adequate to prevent an acute attack [3]. Non-steroidal anti-inflammatory drugs and corticosteroids are used for treating acute inflammation. These drugs have their own limitation from toxicity and longterm safety point of view. Management of chronic gout includes uricostatic, uricosuric and uricolytic groups of drugs. However, use of uricostatic allopurinol gets restricted due to its hypersensitivity reactions, potential drug interactions and difficulty in evaluating the dosage schedule. Uricosuric agents often carry the risk of developing renal stones, while uricolytic drugs are still in the stage of development and available for specific conditions in the form of parenteral administration [4]. It is well-known in Ayurveda medical system that there are numerous medicinal plants, classified under the category of Pashanabheda (stone-dissolving). R. aquatica, A. lanata, B. ligulata are examples of ⁎ Corresponding author. Director Clinical Research & Integrative Medicine, Medical Research Centre, Kasturba Health Society, Sthanakvasi Jain Aradhana Dham, K. Deasi Road, Vileparle, Mumbai-400056, India. Tel.: +91 22 26715147, +91 22 26246119. E-mail address:
[email protected] (A.A. Raut). 0367-326X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.06.001
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clinical usefulness in urolithiasis [5]. Gokshuradi-guggulu, an Ayurvedic formulation, which includes T. terrestris and C. wightii as the principal ingredients, is commonly prescribed for rheumatic conditions as well as for urinary disorders [6]. Commiphora wightii is also a well-known anti-inflammatory agent [7]. Punarnavadi kwath, which contains Boerhaavia diffusa as the main ingredient, is also used for both inflammatory conditions and urinary disorders [8]. Antiinflammatory activity of B. diffusa has been well-documented [9]. In this investigation, the collective traditional knowledge, and clinical experience [10,11] led to the choice of three medicinal plants, viz. B. diffusa, R. aquatica and C. wightii Bhandari syn. C.mukul Engl. for the study of their ability to dissolve in vitro BCP, CPPD and MSUM crystals. 2. Experimental 2.1. Plants B. diffusa L. (Nyctaginaceae), roots obtained from Vasai, Thane district, Maharashtra and the R. aquatica Lour (Boraginaceae), roots were collected from Savantwadi, Maharashtra in the Winter season and authenticated by Dr. M.R. Almeida, Taxonomist, Mumbai. C. wightii Engl. (Burseracae), gum resin was obtained from Barda, Gujarat in Spring and authenticated by Dr. P.S. Nagar, Saurastra Universty, Rajkot. 2.2. Extraction Plants material shade-dried and powdered was Soxhlet extracted with EtOH and aq.EtOH. Moreover, plants material aqueous extract was boiled giving a decoction as per Ayurvedic tradition [12]. The extracts were evaporated to dryness in vacuo and stored at 4 °C. The % and pH of extracts are reported in Table 1 and classes of compounds are reported in Table 2. 2.3. Effect of extracts of crystal dissolution The three extracts, hot EtOH, aqEtOH and aqueous of the three plants were dissolved in their respective solvents. The concentration of the extracts as per the recommended dosage was calculated according to Jukevar et al. [13]. The dissolution of crystals of various shapes and sizes was observed in twenty-four-microwell plates. Each extract (500 µl) was added to 1 mg of crystals in each well and the plates were maintained on a shaker at slow speed for 10 min. [BCP, CPPD and MSUM crystals prepared by single diffusion gel growth technique were obtained from Dr. Mihir Joshi, Department of Physics, Saurashtra University, Rajkot. BCP, CPPD and MSUM crystals were also obtained from Sigma (Catalogue Nos. H2052, C8017, U2875 respectively).] Four different concentrations of each of these extracts were used for the study, the recommended dose, double the recommended dose, half of the recommended dose and quarter of the recommended dose. The calculated recommended concentration for B.diffusa and R.aquatica extracts was 40 µg/ml and for C.wightii ethanolic and hydroethanolic extracts was 150 µg/ml. For C.wightii aqueous extract, it was 50 µg/ml. EtOH, aq.EtOH (1:1 EtOH:water) and distilled water were used as controls. The plates were then incubated at 37 °C and dissolution of the crystals was monitored under the inverted microscope (Carl Zeiss Axiovert 40 CFL) at different time points, and incubation was terminated when none of the crystals was visible, mostly at the end of 24 h. 3. Results MSUM crystals (obtained from both the sources) dissolved completely in the presence of R.aquatica and C.wightii aqueous extracts at the recommended dosage and at double the dosage of the extracts at 24 h (Figs. 1 and 2). No effect Table 1 Percentage extractive values and pH values of plant extracts Material
Hot EtOH
Hot aq EtOH
Hot aqueous
Extractive values (%) B.diffusa root R.aquatica root C.wightii gum resin
5.45 10.09 31.59
6.49 12.27 25.75
Hot EtOH
Hot aqEtOH
Hot aqueous
6.5 6.0 3.5
6.5 6.5 6.0
pH values 10.58 8.97 9.36
6.0 4.0 3.5
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Table 2 Classes of compounds identify in plants extracts Classes of compounds
C. wightii E
Alkaloids Glycosides Tannins/ phenolic compounds Saponins Steroids a b c
− − − − +
a
B. diffusa HE + − – + +
b
AQ + − − + −
c
E + + − − +
a
R. aquatica HE + + − − −
b
AQ + + − − −
c
Ea
HE b
AQ c
− − + − −
− − + − −
− + + − −
Ethanolic extract. Hydroethanolic extract. Aqueous extract.
was seen with B.diffusa. The dissolution took place in stages. In the beginning, the crystals were observed as solid structures. However, after 16 h of incubation, the crystals appeared as spongy particles and finally, at 24 h, the crystals were totally dissolved. Lower dosages of these extracts had no apparent effect on the MSUM crystals. Similarly, no dissolution of either BCP or CPPD crystals was observed in the presence of any of the extracts at all four concentrations. Ethanolic and hydroethanolic extracts of these three plants as well as the solvents kept as controls did not show any crystal dissolution activity. 4. Discussion The observed solubilisation of the crystals in vitro could be dependent on a number of factors. Of these, the solvent is expected to play a very important role [14]. The phosphate crystals, viz. BCP and CPPD crystals are known to be practically insoluble in water, EtOH or acetic acid; similarly, the solubility of urate crystals, viz. MSUM is low in any known solvent [15]. Surprisingly, it is only the aqueous extracts of R. aquatica and C.wightii that dissolved MSUM crystals. One may consider that extremes of pH may influence dissolution of crystals. However, the pH of aqueous extracts of all the three plants is not very acidic in nature; in fact it is near neutral, whereas pH of ethanolic and hydroethanolic extracts is low (Table 1), but has no dissolution activity. An increase in temperature also facilitates dissolution; however, in this case, the experiments were carried out at 37 °C, which is the physiological body temperature. Obviously, it is the constituent chemical composition of the aqueous extracts which played the important role in crystal dissolution. It is interesting to note that only aqueous extracts of R. aquatica and C.wightii were effective against MSUM crystals, but not against phosphate crystals. Joshi et al. have observed the growth inhibition activity of BCP, CPPD and
Fig. 1. Effect of R. aquatica aqueous extract on dissolution of MSUM crystal at 24 h. Legends: a) MSUM + R. aquatica aqueous extract (40 µg/ml)at 0 h (40×) b) At 24 h (40×), showing dissolution of MSUM crystals.
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Fig. 2. Effect of C. wightii aqueous extract on dissolutiom of MSUM crystals at 24 h. Legends: a) MSUM + C. wightii aqueous extract (150 µg/ml) at 0 h (40×) b) At 24 h (40×) ,showing dissolution of MSUM crystals.
MSUM crystals with aqueous extracts of all the three plants by the gel growth technique [Personal communication]. In other words, the effect of the extracts varies according to the model systems; whereas all the aqueous extracts of the three plants have growth inhibition activity, it is only the aqueous extracts of R. aquatica and C. wightii that have the capacity to dissolve already formed crystals. Obviously, the two mechanisms could be different. In view of the aforesaid results, isolation and characterization of the active principles of the plant extracts for crystal dissolution is worth investigation. Acknowledgments The financial support of the Department of Biotechnology (DBT), Government of India, for Research Grant 102/ IFD/SAN/812/2002–2003 is gratefully acknowledged. Our special thanks to the DBT Task Force Expert Committee and especially Prof. Nambiar for suggesting Rotula aquatica for this research work. We are also extremely grateful to Dr. M. R. Almeida (Taxonomist, Mumbai), Dr. J. K. Pathak, Dr. V. Naik (Zandu Pharmaceuticals Ltd., Mumbai) and Dr. P. S. Nagar (Saurashtra University, Rajkot) for their assistance in the collection and authentication of the plants used in the study. We thank the authorities of Bharatiya Vidya Bhavan for encouraging research under the A.I.M.D. project. References [1] Hamilton JA, McCarthy G, Whitty G. Arthritis Res 2001;3:242. [2] Goodman and Gilman. In: Molinoff PB, Ruddon RW, editors. The pharmacological basis of therapeutics, 9th Ed, vol. 1. New York: McGraw-Hill; 1996. p. 647. [3] Kot TV, Day RO, Brooks PM. Med J Aust 1993;159:182. [4] Pande I. Indian J Pharmacol 2006;1:60. [5] Arya Vaidya Sala. Indian med plants: a compendium of 500 species, vol. 5. Hyderabad: Orient Longman; 1996. p. 13. [6] Sharangdharacharya, Madhyamkhanda. In: Shastri P, editor. Sharangdhar Samhita. Varanasi: Chaukhamba Orientalia; 1983. p. 7/ 84–85. [7] R.P. Rastogi, B.N. Mehrotra, Compendium of Indian Medicinal Plants, Vol 2. Lucknow: Central Drug Research Institute and New Delhi: National Institute of Science Communication, 1999. pp. 204. [8] Govinddas Sen. Shothchikitsa. In: Shastri R, Hindi Commentator, Bhaishajya Ratnavali. Varanasi: Chaukhamba Sanskrit Sansthan, 1983. pp. 557. [9] Satyavati GV, Raina MK, Sharma M. Medicinal plants of India, vol. 1. New Delhi: Indian Council of Medical Research; 1976. p. 139. [10] Joshi VS, Joshi MJ. Indian J Phys 2001;75A:159. [11] Joshi VS, Joshi MJ. Cryst Res Technol 2003;38:817. [12] Sharangdharacharya. Madhyamkhanda. In: Tripathi B, Hindi Commentator. Sharangdhar Samhita. Varanasi: Chaukhamba Surbharati Prakashan, 2001. pp. 133/2(1–2). [13] Juvekar AS, Amonkar AJ, Saiki T, D'Cruz AK, Pradhan SP. Cancer Biother Radiopharmac (USA) 1998;13:81. [14] Morrison RT, Boyd RN. Organic chemistry. 6th Ed. New Delhi: Prentice Hall; 1992. p. 249. [15] Merck Index. 9th Ed. Rahway: Merck and Co., Inc.; 1976. p. 8479.
