MATERIALS AND METHODS

All the chemicals, including analytical grade calcium oxalate and solvent were purchased from Sigma Aldrich.

Scanning electron microscope: The CaO_x crystals were analyzed using a VEGA3 TESCAN (Czech Republic) Scanning Electron Microscope (SEM). For the Scanning Electron Microscope (SEM) analysis, samples were coated with a 5 nm-thick gold layer in a sputter coater system. The conductive coating is important because it prevents the charging of the specimen surface during the observation with the SEM, especially when the microscope is operated at high vacuum¹⁴.

Collection of plant material: The Pedalium murex (L.) seeds was collected from Thanjavur, Tamil Nadu, India during the month of May-June, 2017 and it was authenticated by Rev Dr. S. John Britto SJ, Director, The Rapinat Herbarium and Centre for Molecular Systematic, St. Joseph College (Autonomous), Tiruchirapalli, Tamil Nadu, India.

Preparation of extracts: The dried seeds of Pedalium murex were pulverized and extracted using ethanol in Soxhlet apparatus. Ethanol was used for the process as it is safe for ingestion and also can be evaporated after extraction. The extract was concentrated to dryness at room temperature 34°C to get dried extract. The dried extract was diluted with distilled water to 50 g L^–1, which was used for further analysis.

Collection of urine sample: Twenty-four-hour urine specimens were collected from a healthy man, who had no history of kidney stone disease. The samples were refrigerated without preservative for the duration of the collection.

Study without inhibitor (without plant extract): A mixture of 150 mL of urine sample and 50 mL of 0.05 M calcium oxalate was taken in a beaker. After vigorous shaking, the solution was covered with a film and left undisturbed for 48 h. Crystals were centrifuged 3000 rpm for 30 min. The supernatant was discarded and the crystals were transferred to a specimen plate for SEM analysis.

Study with inhibitor (with plant extract): A mixture of 150 mL of urine and 50 mL of 0.05 M calcium oxalate solution was taken in a beaker. After vigorous shaking, the solution was covered with a film and left undisturbed for 48 h. After 48 h, 50 mL of seed extract solution was added in the above mixture. The whole solution was covered with a film and left undisturbed for 24 h. Crystals were centrifuged 3000 rpm for 30 min. The supernatant was discarded and the crystals were transferred to a specimen plate for SEM analysis.

GC-MS analysis: The JEOL GCMATE II GC-MS with data system is a high resolution, double focusing instrument was used to study the presence of phytochemicals in P. murex seeds extract. Ethanol extract was used for the GC-MS analysis.

RESULTS AND DISCUSSION

Crystalline calcium oxalate monohydrate (COM) is the most common material that create kidney stones. The main findings of the present study were that extracts from P. murex seeds inhibited the crystallization of CaO_x in solution. Figure 1a showed maximum number of CaO_x crystals and the CaO_x crystals are easily identified by their heterogeneous shape (Fig. 1b-e).

Fig. 1(a-e):	SEM images of urine sample along with CaOx crystals (without plant extract)

Fig. 2(a-d):	SEM images of urine sample with Pedalim murex extract. Images clearly showed that, in the presence of plant extract, the length and width of the CaOx crystals were reduced, dissociated and dissolved

Figure 1a showed maximum number and largest size of crystals (without extract), while Fig. 2a showed comparatively less number and smaller size of crystals (with extract). There were less and smaller particles with increasing concentrations of extract as shown in various SEM micrographs (Fig. 2a-d). The seed extract had dissociated (Fig. 2b and c) and dissolved (Fig. 2d) the CaO_x crystal growth. The extract of seed causes fewer numbers of crystals in solution (Fig. 2a), thereby reduced super saturation and the size of the particles. This property of the extract is, therefore, advantageous in preventing urinary stone formation by inducing the excretion of small particles from the kidney and reducing the chance of retention in urinary tract. Further, the seeds of Pedalium murex is considered to be diuretic^14-16 and diuretic effects may also reduce stone development when total fluid intake and output increased and such effects have been attributed to several herbal preparations. Very similar results to this study were obtained by Carvalho and Vieira¹⁷. In present study, the crystals were examined optically and with X-ray diffraction and morphology was found to be closely related to relative super saturation.

Plant extracts may contain substances that inhibit the growth of CaO_x crystals. This property of plants may be important in preventing kidney stone formation¹⁸, CaO_x crystals induced by urinary macromolecules was less tightly bound to epithelial cell surfaces, which are then excreted with urine¹⁹. Pedalium murex contains various chemical compounds¹⁵, which themselves possess ability to inhibit the crystallization of calcium oxalate thereby preventing crystal growth and aggregation. Electron microscopic observation revealed that the extract visibly reduced the crystal size with significant decrease in population of CaO_x crystals. These results indicate that the seed extract of P. murex is highly responsible for the inhibition of CaO_x crystal growth. This could be beneficial in the prevention of stone formation. Not only will the formed crystals be smaller, but the implication that these crystals might have a lower likelihood of aggregation shows that the crystals formed could more easily be eliminated by natural means²⁰.

Medicinal plants are used for the prevention of kidney stones from a long time in different countries^21,22.

Fig. 3:	GC-MS chromatogram of seeds of P. murex

Table 1:	Compounds identified in the seeds P. murex

The preventive efficiency of phytochemicals have been evaluated in kidney stones²³. Totally, 9 compounds identified from the GC-MS study (Table 1 and Fig. 3). GC-MS study revealed the presence of oleic acid (classified as a monounsaturated omega-9 fatty acid), phytol, phytosterol, coumarine derivatives etc., (Table 1) may possesses a protective effect against development of calcium stones in the kidneys. This is very beneficial for stone formers since studies suggest that the biochemical pathways of a stone former will always promote stone formation but the presence of growth modifiers such as phytochemicals found in seeds of P. murex could aid in the elimination of stone.

CONCLUSION

It is concluded that Scanning Electron Microscopy (SEM) is a necessary tool to identify and study several renal structures and their behavior as a consequence of the calculi removal, so that it is possible to supply important information for the surgical ways of intervention in order to prevent a relapse. The P. murex seed extract decreased the crystal size and inhibited crystal aggregation. The seed contains good amount of fatty acid and several phytochemicals. This study may suggest that P. murex can be used as therapeutic agent for the treatment of urinary calculi or its prevention.

SIGNIFICANCE STATEMENT

Kidney stone disease is a common chronic disorder seen in humans and the most common type of renal stone is made of calcium oxalate. Kidney stones cause serious health problems such as severe pain, urinary tract obstruction and infection that adversely affect comfort of individuals. Pedalium murex Linn. is an important drug widely used in India for the patients of urolithiasis. Scanning Electron Microscopy (SEM) can provide degradation/dissolution information of urinary calculi. Common methods of kidney stone analysis, such as light microscopy and FTIR, provide limited information of the stones. SEM gives high spatial resolution (~1 nm). SEM can provide degradation/dissolution information of urinary calculi. This study may suggest that Pedalium murex seeds and its phytochemicals can be used as therapeutic agent for the treatment of urinary stones or their prevention. This investigation will helpful in developing new drugs for the treatment of kidney stones, with therapeutic and economical value in the future.

ACKNOWLEDGMENT

Authors thank Periyar TBI, Periyar Maniammai University, Thanjavur for SEM analysis.