Chapter 1 INTRODUCTION

Chapter 1 INTRODUCTION 1.1 CONTEXT OF THE STUDY 1.2 RELEVANCE OF ETHNOBOTANICAL STUDIES IN DRUG DISCOVERY 1.3 NEED AND SIGNIFICANCE OF THE PRESENT STUDY 1.4 HYPOTHESIS OF THE STUDY 1.5 OBJECTIVES OF THE STUDY 1.6 MATERIALS AND METHODS IN BRIEF 1.7 SCOPE OF THE STUDY 1.8 FORMAT OF THE REPORT

Chapter 1 INTRODUCTION 1.1 Context of the Study Human race is constantly being challenged by many dreadful diseases and it is an uphill task to combat them in the present scenario. Leading healthy life is a challenge in a highly competitive world, where people hardly get a chance to care about their diet or assure the quality of the food they eat. The Cultural Revolution and luxurious life styles have made remarkable shift in the food habits of people all over the world with increased reliance on junk foods and soft drinks. This situation has brought about an alarming situation with sporadic increase in the incidences of life style diseases such as diabetes mellitus, heart diseases, arthritis, cancer and urolithiasis, all over the world. Urolithiasis is one among the oldest and widely reported diseases known to mankind. The earliest reports of urinary calculi have been found in the tombs of Egyptian mummies dating back to 4000 BC (Eknoyan, 2004). It is ubiquitous and is prevalent in relatively young and productive age group. It carries significant morbidity and imposes tremendous financial burden on healthcare system. With changing lifestyles and climate, its prevalence has shown a 37% rise in USA from 1976 to 1994 (Ngo and Assimos, 2007). Life-time risk of urolithiasis varies from 1-5% in Asia, 5-9% in Europe, 10-15% in USA and 20-25% in middle-east; lowest prevalence is reported from Greenland and Japan (Bartoletti et al., 2007). In India, the situation is quite alarming, and about 12 percent of people were expected to have urinary stones, out of which 50 percent may end up with loss of kidneys and renal damage (Tribuneindia census, 2000). The above situation reminds us the importance of research inputs to develop suitable treatment strategies for urolithiasis. The reasons for kidney stone formation or urolithiasis are multifold such as life style, dietary habits, family history and global warming. Majority of kidney stones are composed of calcium oxalate and phosphate crystals (~80%). The rest 2

are composed of uric acid (5-10%) and struvite crystals (5-15%). Other rare constituents are cystine, xanthine, dihydroxy-adenosine and various drugs (Moe, 2006). Kidney stone formation is a complex process that results from a series of several physico-chemical events including supersaturation, nucleation, growth, aggregation and retention of urinary stone constituents within the renal tubules. Three distinct stages can be recognized in the process of stone formation. The first stage involves crystal nucleation, growth and aggregation. In the second stage, crystals are retained within the kidneys, renal tubules and/or interstitium, and in the final stage, retained crystals move from inside of the kidney to the renal papillary surface to form a stone nidus. Minerals in the urine especially calcium, then build on the speck in a similar way to that in which a pearl grows in an oyster shell. The formation of the nidus may be analogous to the first stage in physiologic calcification of bone in which a nucleus of calcium phosphate develops in an organic matrix (Thomas and Howard, 1959; Boyce and King, 1963; Howard et al., 1967; Boyce, 1968). Alternatively, the nidus may form spontaneously by precipitation from supersaturated urine (Vermeulen and Lyon, 1968). It is reported that calcium phosphate is the major solute component which crystallize rapidly in urine. Its presence is also reported in small quantities in renal and urethral stones (Grases, 1993; Tiselius, 1996). Moreover, small amounts of calcium phosphate were detected in the assumed attachment part of the stone (de Bruijn, 1995; Trinchieri, 2006; Miller, 2007). These observations suggest that other stones have their origin on a calcium phosphate precipitate and that the early stages of these stones are attached to the renal papilla (Matlaga et al., 2006; Evan, 2009; Coe, 2010). Once the crystal nidus has been established, it develops into a renal stone by precipitation or by the process of crystal growth. Initially, kidney stones often do not cause any symptoms. Usually, the first symptom of a kidney stone is extreme pain, which occurs when a stone acutely blocks the flow of urine. The pain often begins suddenly when a stone moves in the urinary tract, causing irritation or blockage. If the stone is too large, it often leads to bleeding and blood may appear in the urine. 3

Treatment options depend on the size and nature of the kidney stone. Stones which are smaller than 5mm have a high probability of spontaneous passage which can take up to 40 days (Coll et al., 2002). During this watchful waiting period, patients can be treated with hydration and pain medications. However, stones larger than 5mm or stones that fail to pass are treated by interventional procedures such as ESWEL and ureteroscopy (Knoll, 2007; Pearle et al., 2001). However, these techniques are not free from side effects (Silberstein et al., 2008; Krambeck et al., 2006). Effective kidney stone prevention is dependent on the stone type and the detection of risk factors for stone formation. An individualized treatment plan incorporating dietary changes, supplements, and medications can be developed to assist or prevent the formation of new stones. Without considering the underlying etiology of the stone disease, patients should be instructed to increase their fluid intake, minimize sodium and animal proteins in their diet (Park and Pearle, 2007). It is reported that high protein intake reduces urine ph and citrate and enhances urinary calcium excretion via bone re-sorption and reduces renal calcium reabsorption. Stone formers are not advised to restrict calcium unless it has been shown that they have an excessive intake of calcium (Tiselius, 1996). A reduced intake of calcium leads to an increased intestinal absorption of oxalate, which itself may account for an increased risk of stone formation. Vitamin C has been implicated in stone formation because of in vivo conversion of ascorbic acid to oxalate. Therefore, a limitation of vitamin C supplementation to 500 mg/day or less is recommended (Park and Pearle, 2007). When dietary modification is inadequate, the next option is to initiate pharmacological treatment. The most effective hypocalciuric agents are thiazide diuretics with hypo-calciuric action, which enhance calcium re-absorption in the distal renal tubules (Laerum and Larsen, 1984). However, its long term use in about 50% of patients is limited because of side effects including fatigue, dizziness, impotence, musculoskeletal symptoms, or gastrointestinal complaints (Park and Pearle, 2007). Another complication is thiazide-induced potassium depletion, which causes intracellular acidosis and can 4

lead to hypokalemia and hypocitraturia (Moe, 2006). Potassium citrate is reported to be effective in the treatment of patients who have calcium stones and normal urinary calcium. By providing an alkali load, potassium citrate increases urinary ph and citrate, thereby mediating the inhibitory effects of macromolecular modulators of calcium oxalate crystallization (Mattle and Hess, 2005). The major drawback for a more widespread use of alkali citrate preparations is the relatively low tolerability of available alkali citrate preparations. Adverse effects that reduce treatment compliance have been noted mainly in the gastrointestinal tract and include eructation, swelling and diarrhoea (Mattle and Hess, 2005). In conclusion, the detailed review of literature revealed that none of the above listed treatment modalities provide satisfactory treatment options for urolithiasis. The above situation necessitates the need to develop novel drugs for urolithiasis based on ethnobotanical approaches in drug discovery. 1.2 Relevance of Ethnobotanical studies in drug discovery Over the last century, ethnobotany has evolved into a scientific discipline that focuses on the people-plant relationship in a multidisciplinary manner. According to Schultes (1992), the relationship between plants and human cultures is not limited for food, clothing and shelter alone but also includes religious ceremonies, ornamentation and health care. As Balick (1996) rightly pointed out, it has become increasingly valuable in the development of health care and conservation programs in different parts of the world. According to the reports of WHO, 70% - 95% of citizens in majority of the developing countries still rely on traditional medicine as their primary source of medication (Robinson and Zhang, 2011). They rely on medicinal plants because of their effectiveness, cultural preferences and lack of modern healthcare alternatives (Caniago and Siebert, 1998). The ethnobotanical approach can offer strong clues regarding the biological activities of many indigenous plants (Cox and Balick, 1994). In the above context, the ethnobotanical studies conducted by earlier researchers have made remarkable contributions to bring out medicinal virtue/therapeutic potential of many indigenous plants discovered by traditional healers. Drugs like Aspirine, Codeine, Ipecac, Pseudoephedrine, Quinine, Reserpine, Scopalamine, Theophyline and 5

Vinblastin are a few such potential chemical constituents developed in the past based on such ethnobotanical investigations. According to Principe (2005), about 25 percent of the medical drugs in the developed countries are made either from plants or their derivatives. It is estimated that less than 10% of the world s genetic resources have been studied seriously as sources of medicine. Yet, a substantial number of drugs have been developed from these plants which are active against a number of diseases (Fabricant and Farnsworth, 2001). In short, Traditional Botanical Knowledge (TBK) has helped man to isolate medicines for a large number of diseases and will continue to be a promising source for further research in drug development. In the traditional system of medicine, Pashanabheda/Asmabheda group of plants are often prescribed as diuretic and antiurolithiatic drugs. The word Pashanabheda/Asmabheda literally means one that breaks stone (Pashana /Asma = stone, bheda = that which breaks) preferably, with reference to urinary stones in the body. There are about 40 sanskrit synonyms attributed to Pashanabheda in different Ayurvedic texts. Presently, more than seven medicinal plants like Bergenia ligulata, Aerva lanata, Tribulus terrestris, Ammania baccifera, Nothosasrava brachiata, Homonoia riparia, Rotula aquatica, Coleus aromaticus, Bryophyllum calicynum and Didymocarpus pedicllata are being used either as genuine or as substitute drugs across India (Mukhopadhyaya, 1929; Pandey, 2002; Venugopal, 2009). However, no serious studies have so far been taken up to evaluate and document the traditional botanical knowledge of antiurolithiatic plants in Kerala state. Hence, an earnest attempt is made in the present investigation to identify indigenous antiurolithiatic medicinal plants and subject them to crystallographic evaluation. 1.3 Need and significance of the present study The currently employed medical management of urinary calculi includes lithotripsy and surgical procedures. These treatment options are prohibitively expensive for the common man, and with these procedures recurrence is quite common and the patient has to be subjected to careful follow up for several years (Christina, 2002). As pointed out by Miller et al. (2007), there are no satisfactory 6

drugs in modern medicine which can dissolve the urinary stone and patients mostly rely on alternative systems of medicine for better relief. This necessitated the need for further research on crystallization of urinary stone constituents giving due emphasis for the development of novel in vitro dissolution studies/ strategies for the prevention and treatment of kidney stone disease. In this respect, it is to be noted that only limited attempts have been made on in vitro crystallization, growth and dissolution studies of CHPD crystals utilizing bioactive compounds of indigenous medicinal plants. It is expected that traditional botanical knowledge on medicinal plants and screening their efficacy by developing novel experimental strategies incorporating the emerging tools and techniques in phytochemistry and crystallography would help us to generate additional knowledge, provide a better explanation, placement and direction for further research in the area of urolithiasis. The present study is undertaken in the above context, and is entitled: ETHNOBOTANICAL AND CRYSTALLOGRAPHIC STUDIES OF SELECTED ANTIUROLITHIATIC MEDICINAL PLANTS. 1.4 Hypothesis of the study Present study was designed to test the following hypothesis: Traditional practitioners and herbal collectors may have sufficient knowledge about potential antiurolithiatic medicinal plants and their indigenous uses/ preparations. Gel method of crystallization is an effective strategy for growth dissolution studies of Calcium Hydrogen Phosphate Dihydrare (CHPD) crystals. The aqueous extract of Rotula aquatica Lour (Kalloorvanchi), Aerva lanata(l)juss. ex Schult (Cherula), Ensete superbum (Roxb.) Cheesman (Kalluvazha), Achyranthes aspera L. (Vankadaladi) and Spheranthus indicus L. (Adakkamaniyan) may have the potential to bring out morphological and structural changes in the CHPD under in vitro growth conditions. 7

1.5 Objectives of the study The major objectives of the present study are: 1. To document the Traditional Botanical Knowledge (TBK) of antiurolithiatic medicinal plants from Traditional healers and Herbal collectors of Kottayam district, Kerala, India. 2. To standardize the growth of CHPD crystals in metasilicate gel medium. 3. To study the efficacy of Rotula aquatica Lour, Aerva lanata (L) Juss. ex Schult, Ensete superbum (Roxb.) Cheesman, Achyranthes aspera L. and Spheranthus indicus L. on in vitro growth inhibition of CHPD. 4. To characterize the gel grown CHPD crystals by FTIR, XRD, TGA/DTA and SEM / EDX. 1.6 Materials and Methods in brief An ethnobotanical survey was conducted among the traditional practitioners and herbal collectors of Kottayam district to document the traditional botanical knowledge regarding antiurolithiatic medicinal plants. The plant specimens were collected and identified using relevant taxonomic literature. Selected plants were subjected to further studies based on crystallographic techniques. Modified single diffusion gel growth technique in sodium metasilicate gel was employed for screening the antilithic property of the selected medicinal plants. The crystallization and growth inhibition of CHPD crystals were done using the aqueous extract of selected medicinal plants. The gel grown crystals were then characterized by FTIR, XRD, TG/DTA and SEM/EDX. 1.7 Scope of the study Traditional medicines and practices are handed down through the generations mostly by Word of Mouth. Documenting the indigenous knowledge through ethnobotanical studies is important for the conservation of biological resources as well as their sustainable utilization. It is expected that the traditional botanical knowledge would be helpful for the preparation of a catalogue of traditionally used antiurolithiatic medicinal plants. The study has tremendous 8

scope to evaluate the efficacy of many herbal based drugs and phytoactive compounds of yet unexplored medicinal plants to suppress crystallization of calcium phosphate, calcium oxalate and related compounds that cause urolithiasis. The present study has tremendous socio-political and economic significance in the context of the emerging question of safe guarding of patent and proprietary rights of the traditional medicine especially in the light of IPR Act in vogue. 1.8 Format of the report The report is presented in 5 chapters and the details being as follows: Chapter 1 is introduction and as noted above contains all relevant sections of an introductory chapter, such as a brief account of context of the study, Calcium Hydrogen Phosphate Dihydrate (CHPD), treatment and prevention of kidney stone disease, ethnobotany and its relevance in drug development, need and significance of the present study, objectives of the study, hypothesis of the study, materials and methods in brief, scope of the study and format of the report. Chapter 2 presents the major conclusions that have emerged from the review and appraisal of the literature pertaining to the area under investigation. An earnest attempt is made in this chapter to review almost all aspects of urolithiasis such as: history of urolithiasis in global and Indian context, current scenario of urolithiasis, Ethnobotany and its relevance in identifying antiurolithiatic medicinal plants, Traditionally used antiurolithiatic medicinal plants, In vitro and in vivo antiurolithiatic studies using medicinal plants, anti urolithiatic phytoconstituents, risk factor of kidney stone disease, pathogenesis of renal stone and urinary stone types. A brief account of Calcium Hydrogen Phosphate Dihydrate (CHPD), its growth behavior and influence of additives on growth kinetics is also included. Crystal growth by sodium metasilicate gel method and characterization of crystals by X-ray powder Diffraction (XRD), Energy Dispersive X-Ray analysis (EDX), Thermal analysis (TGA/DTA), Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) were also presented in detail. 9

An elaborate review of plants selected for the present in vitro antiurolithiatic investigation viz. Rotula aquatica Lour, Aerva lanata (L.) Juss. ex Schult., Ensete superbum (Roxb.) Cheesman., Achyranthes aspera L. and Spheranthus indicus L., were also attempted in this chapter. Chapter 3 gives a brief account of materials and methods employed for the ethnobotanical as well as growth inhibition studies on CHPD by plant additives. A schematic representation of the experimental strategy is also included in this section. Chapter 4 gives the major results emerged from the Ethnobotanical investigations of antiurolithiatic medicinal plants. It also provides valuable results from crystallographic investigations based on growth inhibition of CHPD conducted with the aqueous extract of 5 medicinal plants viz. Rotula aquatica Lour, Aerva lanata (L.) Juss. ex Schult., Ensete superbum (Roxb.) Cheesman., Achyranthes aspera L. and Spheranthus indicus L. An attempt is also made to substantiate the results based on relevant literature. Chapter 5 summarizes the study in retrospect, major results, scope and implications of the study. 10