In vitro Growth and Inhibition Studies of Monosodium Urate Monohydrate Crystals by Different Herbal Extracts

Problem statement: A large number of people in the world have been suf fering from gout for centuries. Gout is the term given to a metaboli c condition in which the signs and symptoms results from the deposition of crystals of Monosodium Urate Monohydrate (MSUM) in various connective tissues. Approach: It was quite difficult to grow good quality large M SUM crystals because uric acid was having very low solubility in any known solvent s. Results: Attempts were made in the present investigation to grow MSUM crystals in vitro by single diffusion gel growth technique, which is quite suitable to mimic the growth of MSUM crystals in vivo up to a certain extent. Good quality needle type crystals were grown in the gel, which were characte ized by FT-IR, Powder X-ray diffraction and Thermo-gravimetry. The characterization study confi rmed the formation of MSUM crystals in the experiments. Herbal extract solutions of Boswellia serrata Linn., Aerva lanata juss ex. Schult, Routula aquatica Lour. and Boerhavia diffusa Linn., were used for the growth inhibition study of MSUM crystals. Aqueous herbal extract solutions wer e poured as supernatant solutions along with the control solution on the set gels during the crystal growth experiment. The growth observations and the measurements of crystal dimensions using optical mi croscope suggested that Routula aquatica Lour. and Aerva lanata juss ex. Schult extracts exhibited good crystal growth inhibition results. Conclusion: Routula aquatica Lour. extract dissolved MSUM crystals after 15 days of po uring the supernatant solution on the gel. This in vitro study may be helpful for in vivo studies, which may further lead to develop a preclinical formulation f or gout treatment.


INTRODUCTION
Gout is the term given to a group of metabolic conditions, in which the signs and symptoms results from the deposition of crystals of Monosodium Urate Monohydrate (MSUM) in various connective tissues and joints. It is also an auto inflammatory disorder associated with deposition of MSUM crystals in joints and periarticular tissues. The recent advances, however, suggest that the innate immune system may drive the gouty inflammatory response to MSUM [1,2] . The deposition of these crystals, result from the raised levels of uric acid in blood (hyperuricemia) and various body fluids. Hyperuricemia is not essential requirement of diagnosis of gout, but the risk of gout and its presence increases with the degree and duration of hyperuricemia. The risk factor of gout is also associated with the uric acid and urate compound nephropathy [3] . Nephropathy of uric acid and urate substances is associated with the formation of Monosodium Urate (MSU), ammonium urate and uric acid in urinary tract system. The MSU is rarely observed in renal calculi in spite of human urine often being supersaturated with monosodium urate [4][5][6] . Only exceptionally pure monosodium urate calculi have been described and these were of vesical origin [7] . Hyperuricosuria, simulated by the addition of dissolved sodium urate, promotes the crystallization of Calcium Oxalate. It could also act as an effective promoter of calcium oxalate crystallization according to in vitro experiments [8,9] .
A very few attempts have been made to grow the uric acid or urate crystals. Irusan et al. [10] have reported the growth of ammonium urate crystals using the gel growth technique, whereas Kalkura et al. [11] have reported the growth of spherulites of monosodium urate monohydrate and Kalkura et al. [12] have studied crystallization of pure uric acid.
From this description of accumulated knowledge about the role of Monosodium Urate Monohydrate (MSUM) crystals in gout or gouty arthritis as well as urolithiasis, it is evident that till today there is no sufficient information available to clarify all aspects related to crystallization behavior under physiological conditions and its different characteristics. It is, therefore, important to grow and characterize MSUM crystals and study the inhibitive effect of various herbal extracts on its growth. The growth and inhibition study provides very useful information regarding the potent herbal extracts or herbal formulations, which can inhibit the growth of crystals in vitro and may be useful in prevention or cure of this crystal induce ailments.

MATERIALS AND METHODS
The growth of MSUM crystals is not widely reported. In the present investigation, glass test-tubes of 25 mm diameter and 150 mm length were used as crystal growth apparatus. Sodium metasilicate solution of 1.05 specific gravity and 0.2 M, NaOH solutions were added in equal amounts. This mixture was acidified by 2N, acetic acid in such manner that the pH values within 4.5-5.0 are obtained. This mixture was poured in equal volumes in different test tubes and allowed to set into the gel form. Within 48 h, good quality gels were set. After setting the gel, the 0.07 M uric acid solution was poured gently on that. Good quality needle shaped crystals were grown, which are shown in Fig. 1  material in the ratio 1:8, which was heated in a boiling water bath until the kwath reduces in half of the original volume. All extracts were air dried in a rotary vacuum evaporator to a syrupy consistency and then in a steam bath to thick, pasty consistency. All extracts were stored in glass vials kept in air tight plastic boxes at-20°C. Figure 1 shows the grown crystals. The crystals have been characterized by different techniques.
FT-IR spectrum was recorded on Shimadzu 8400 set up in 400-4000 cm −1 range using powdered samples in KBr medium. Powder XRD was carried out on Philips X'pert using Cu K α radiation. Thermogram was recorded on Pyrif 1, Perkin Elmer, in atmosphere of air, from room temperature to 900°C at a heating rate of 15°C min −1 .

RESULTS
The molecular structure of MSUM is as Shown in Fig. 2. Figure 3 shows FT-IR spectrum of MSUM crystals. Table 1 gives assignments to the peaks.
From the FT-IR spectrum one can confirm the presence of keton group (C = O), carbon-nitrogen bond (C-N), N-H stretching and rocking as well as the presence of water of hydration in the sample. Figure 4 is the powder XRD pattern and Table 2 gives the data. The samples are highly crystalline in nature. Figure 5 is the thermo-gram of MSUM, which shows that the compounds is stable up to 100°C and slowly give up water of hydration and on wards 170°C starts losing crystalline water immediately. The associated water molecule is completely removed at 240°C and becomes anhydrous. Subsequently, when the temperature is further increased, the mass loss occurs very sharply between 370-480°C with the release of gases like hydrogen cyanide, carbon dioxide and carbon monoxide. This is followed by a slow process between 480-750°C with loss of nearly 75-80% of original mass.

Fig. 4: Powder X-ray diffractogram of the MSUM crystals
Finally, it is converted into Na 2 O, which remains stable up to the end of the analysis. One water molecule is found to be associated with the crystals. However, no work is reported on the in vitro growth and inhibition study of MSUM crystals. Figure  6 shows the test tubes having the mixture of uric acid and solution of herbal extracts, which were used for inhibition study. In the case of pure uric acid the average length of grown crystals was 0.675 cm. But for the B. serrata, containing supernatant solution, no considerable change was observed. The same effect was observed in the case of B. diffusa.  Result of inhibition study of MSUM crystals in terms of observed crystal length after seven days of pouring the herbal extract solutions is shown in Table 3. From the Table 3, one can easily conclude that R. aquatica and A. lanata give comparatively good inhibition than the others. However, the ethanolic herbal extract solutions almost gave the same results. As uric acid is completely insoluble in ethanol, it was observed that by adding ethanolic extract the reprecipitation occurred in the supernatant solution. But aqueous extract of B. diffusa (80 mg mL −1 ) was found mildly active. These results were found to be encouraging for the in vivo studies and the formulation of the drug. This study may be helpful to design the therapies for the prevention and cure of gout.

DISCUSSION
Monosodium urate (MSU, NaHC 5 H 2 N 4 O 3 ) is a white powder and soluble in water to the extent up to 120 mg dL −1 [13] , for the sake of comparison a much more limited solubility of uric acid in water is reported [14] , which is only 6.5 mg dL −1 . Allen et al. [15] have found that urate solubility in solutions of 140 mEq L −1 sodium content is temperature dependent, with a two fold drop in solubility between 37 and 25°C. Moreover, Wilcox et al. [6] have carefully reinvestigated the solubility of uric acid and monosodium urate and have found that both are dependent on pH. Uric acid is more soluble with increasing pH, whereas the MSU is less soluble. The solubility of urate in plasma is somewhat greater than the saturation value in aqueous solutions of 0.13 M sodium. Actual determination of solubility of monosodium urate in human plasma (or serum) indicates that the saturation occurs at concentrations about 7 mg dL −1 [16][17][18] . Monosodium urate mainly forms due to the reaction between the Na + ion and the urate ion of uric acid. Due to limited solubility of uric acid in any known solvent it is difficult to grow MSUM crystals.
Grases et al. [19] have studied the structure of urate renal calculi after precipitation of ammonium and sodium urate from the synthetic urine; on the other hand, Grover et al. [20] have studied the in vitro growth of calcium oxalate crystals in the presence of preincubated seed crystals of uric acid and MSU with undiluted human urine.
FT-IR spectroscopy is an excellent tool to identify various chemical bonds in a compound. Kalkura et al. [11] have reported the IR spectra of spherulites of monosodium urate monohydrate. The FTIR spectroscopic study has proved the presence of all functional groups and bonds in MSUM crystals.
Howell et al. [21] have reported the powder XRD patterns and d values of uric acid, MSUM and Disodium Urate Dihydrate (DSUD), but unit cell parameters were not reported. The Powder XRD of tophaceous deposits in gout also pin points MSUM as the prime accuse. It is very difficult to obtain the single crystal XRD data on the present MSUM crystals because they get degraded immediately upon removal from the test tube upon exposure of the atmosphere. However, the present powder XRD study suggests that samples are crystalline in nature.
Thermogravimetric Analysis (TGA) is performed to assess the thermal stability of the substance. Earlier, Schneitzler et al. [22] have reported TGA of purine derivatives such as aminophylline, theoophylline, caffeine and uric acid. It was found that uric acid was stable up to 375°C and thereafter the mass loss occurred through rapid process. However, the MSUM crystals having one water molecule associated with it and became anhydrous at 240°C and above 370°C the mass loss became very rapid. The stabilities of uric acid and MSUM are comparable.
The growth and inhibition study of various biomaterial crystals, particularly, responsible for ailments related to urinary stones were studied. Joshi et al. [23] reported the effect of herbal extracts on the growth of calcium oxalate as well as struvite type urinary crystals by Chauhan et al. [24] . Recently, Parekh et al. [25] have reported the growth inhibition of hydroxyapatite crystals by herbal extracts. From the Table 3, one can easily notce that R. aquatica and A. lanata give comparatively good inhibition than the others. However, the ethanolic herbal extract solutions almost gave the same results. As uric acid is completely insoluble in ethanol, it was observed that by adding ethanolic extract the re-precipitation occurred in the supernatant solution. But aqueous extract of B. diffusa (80 mg mL −1 ) was found mildly active. These results were found to be encouraging for the in vivo studies and the formulation of the drug. This study may be helpful to design the therapies for the prevention and cure of gout.

CONCLUSION
The growth of MSUM crystals was carried out by using single diffusion gel growth technique, which is well suited to mimic the conditions of the growth of crystals in vivo. Very thin, transparent, needle type crystals are observed near the gel-liquid interface. FT-IR spectrum of MSUM crystals confirms the presence of a water molecule, metal-oxygen bond, O-H stretching, N-H rocking and stretching, C = C bond, C = O carbonyl group (keton group) and C-N bond. The powder XRD results confirm the crystalline nature of the sample. MSUM crystals are thermally unstable and finally decompose into Na 2 O. One water molecule is associated with the crystal. . In the case of 1% aqueous extract solutions of B. serrata and B. diffusa the inhibition was not significant, but in the case of 1% aqueous extract solutions of R. aquatica and A. lanata, good amount of inhibition was shown. Till today there is no major remedy is available to dissolve the MSUM crystal in synovial fluid, which is responsible for gout. This study may help the prevention or cure of gout.