In vitro evaluation of anti-neprolithiatic activity of leaves and seeds of Macrotyloma uniflorum on dissolution or removal of kidney stones

Amritan Kachru¹, Monika Bisht¹, Mamta Baunthiyal²*

¹M. Tech Scholar, G.B. Pant Engineering College, Pauri Garhwal, Uttarakhand

²Associate Professor, G.B. Pant Engineering College, Pauri Garhwal, Uttarakhand

*Corresponding Author E-mail: mamtabaunthiyal@yahoo.co.in

ABSTRACT:

Nephrolithiasis is a chronic disease and in medical terms refers to formation of stones in kidneys. A renal calculus or kidney stone is a solid aggregation formed in kidneys from various minerals present in the diet. The kidney stones differ in sizes, shape and types. Kidney stone formation is a complex process that involves nucleation, supersaturation, growth, aggregation and retention within the kidney. The treatment of removal of kidney stones includes Extra corporeal Shock Wave Lithotripsy which can also sometimes cause renal injury. Various plant species have been used for the treatment of kidney stones. Macrotyloma uniflorum (Horse gram) is a popular pulse of Uttarakhand, locally known as Gaheth. M. uniflorum seeds are rich in protein, consumed in majority by poorest section of the society and are cultivated in India since prehistoric times. M. uniflorum seeds have been used by people of Uttarakhand for the treatment of kidney stones though till date no scientific explanation regarding its mechanism of action is known. The leaves have been used as a source of animal feed and are not consumed by humans. In the present study we focus on the comparative anti-nephrolithiatic activity of seeds and leaves of M. uniflorum and also the different bioactive components that may be responsible for dissolution of experimental kidney stones.

KEYWORDS: Anti-nephrolithiatic, Calcium oxalate, kidney stones, Macrotyloma uniflorum, phytochemical test, GC-MS analysis

1. INTRODUCTION:

Kidney stones are solid particles that are formed in the urinary tract. The stones vary in size and smaller ones can pass out of the body without any problems. There is often excruciating pain which results due to blockage of flow of urine by the kidney stones and therefore prompt medical treatment may be needed. Even after the treatment which may involve expensive surgeries, there is a possibility of recurrence of kidney stones formation.

Calcium containing stones, especially calcium oxalate monohydrate, calcium oxalate dihydrate and basic calcium phosphate are the most commonly occurring ones to an extent of 75-90% that is followed by magnesium ammonium phosphate (Struvite) to an extent of 10- 15%, uric acid 3-10% and cystine 0.5-1%. In most of the cases the commonly occurring stones are calcium oxalate or magnesium ammonium phosphate type.

Plant and plant based drugs have efficient pharmacological action and potent effects on body. Macrotyloma uniflorum is a leguminous plant belonging to the family: Fabaceae (alt. Leguminosae). They are annual with cylindrical stems. The pod is slightly curved and smooth, it is an excellent source of iron and molybdenum and proteins¹. The seeds are ovoid and color differs from light red, brown, or black sometimes with small, scattered black spots². This plant has the greatest potential to be used as a source of nutrition, forage and food for malnourished and also in the drought prone areas of the world. It is native to most parts of India and is found up to altitudes of 1000 m. The present study has been undertaken to evaluate different leaves and seeds extracts of M. uniflorum and cystone as a standard for their possible potential to dissolve experimental kidney stones using a modified in vitro model and to characterize the chemical constituents responsible for the activity. Leaves and seeds extracts were prepared in benzene, chloroform, ethanol, ethyl acetate and methanol. Qualitative analysis was done for detection of various phytochemicals and these extracts were used along with the standard cystone drug to evaluate dissolution of experimental kidney stones. The gas chromatography mass spectroscopy analysis (GC-MS) was also done to identify various bioactive compounds that might have anti-nephrolithiatic activity.

2. MATERIALS AND METHODS:

2.1. Collection of sample:

M. uniflorum seeds were collected and stored in air tight container. The seeds were sown and grown in campus field. The leaves were collected and washed with distilled water. Both seeds and leaves were taken for further analysis.

2.2 Extraction and Isolation:

The leaves and seeds were grinded in a blender and about 20 gms of powder was extracted with benzene, chloroform, ethanol, ethyl acetate and methanol in soxhlet ⁸. All extracts were concentrated on water bath. All the prepared extracts were stored in reagent bottles and these were subjected to qualitative chemical tests to detect the presence of different classes of phyto constituents.

2.3. Phytochemical analysis³

The extracts were analyzed for different phytochemicals present in M. uniflorum that included test for the presence of alkaloids, glycosides, flavonoids, proteins, phenols, reducing sugars, saponins, steroids, tannins and triterpenoids.

2.4. Evaluation for Anti-nephrolithiatic Activity⁴

2.4.1 Preparation of experimental kidney stones (Calcium oxalate stones) by homogenous precipitation

Equimolar solution of calcium chloride dihydrate was dissolved in distilled water and sodium oxalate in 10ml of 2N H₂SO₄ were allowed to react in sufficient quantity of distilled water in a beaker. The resulting precipitate was calcium oxalate. The precipitate was washed with distilled water and dried at 60 ºC for 4 hours.

2.4.2 Preparation of semi-permeable membrane from farm eggs

The semi-permeable membrane of eggs is in between the outer calcified shell and the inner contents like albumin and yolk. The shell was removed chemically by placing the eggs in 2M HCl for an overnight. This resulted in the complete decalcification of the egg and then it was washed with distilled water. Carefully with a sharp pointer a hole was made on the top so that the contents squeeze out completely from the decalcified egg. The membrane was rinsed with distilled water and stored in refrigerator.

2.4.3 Estimation of Calcium oxalate by Titrimetry

1mg of sample and 10mg of the extract/ compound/ standard and were packed together in semi permeable membrane. The membranes were suspended in conical flasks containing 100ml of 0.1 M TRIS buffer. 1mg of calcium oxalate served as a negative control. The conical flasks of all groups were placed in a preheated incubator for about 7-8 hours. The contents of semi-permeable membrane from each group were removed into a test tube. 2 ml of 1 N sulphuric acid was added and titrated with 0.9494 N KMnO₄ till a light pink color end point was obtained.1ml of 0.9494 N KMnO₄ is equivalent to 0.1898 mg of calcium. The amount of undissolved calcium oxalate was subtracted from the total quantity used in the experiment in the beginning to determine how much quantity of calcium oxalate actually test substance(s) could dissolve.

2.5. GC-MS Analysis

The methanolic extract was directly used for the analysis. GC-MS was carried out on a GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan) system with head space sampler (AOC-20s) and auto injector (AOC-20i) that was equipped with mass selective detector, having ion source temperature of 230°C, interface temperature of 260°C, a solvent cut time of 2.50 min threshold of 1,000 eV and mass range of 40 to 650m/z. The compounds were separated using a Rtx 5 MS capillary column (Restek Company, Bellefonte, USA: cross bond 5% diphenyl /95% dimethyl polysiloxane) having dimensions 30 m (length) × 0.25 mm(diameter) × 0.25 μm (film thickness). The temperature of the injector was initialized to 250°C, having a split injection mode. The temperature of GC-MS was programmed from 100°C (3 min) that was then further increased to 280°C at a ramp rate of 10°C/min (19 min hold). The carrier gas was helium at a linear flow velocity of 40.9 cm/s. The debit of gas (helium) vector was fixed to 16.3 mL/min, with a total flow of 1.21 mL/min. The volume of injected sample was 1.5 mL of methanol extract. The components present in the sample were identified by comparison of their retention indices (RI) relative to homologous alkane series (purchased from Sigma, St. Louis, USA) and by comparison of their mass spectral fragmentation patterns with those data provided in WILEY8.LIB, NIST08.LIB, NIST08s.LIB and NIST.LIB. The identification of compounds was assumed when a good match of mass spectrum and RI was achieved.

3. RESULTS AND DISCUSSIONS:

The qualitative analysis showed presence of proteins, flavonoids, phenols, triterpenoids, glycosides, tannins and sterols in different extracts of seeds and leaves of M. uniflorum that could play a major role in its anti-nephrolithiatic activity (Table 1).

Glycosides are a class of medications used to treat heart failure. Phenols had already been reported to have antioxidant properties. The phenols prevent calcium oxalate formation and their deposition by scavenging of free radicals. Tannins have antioxidant properties and it effectively blocks renal calcification⁵. It reduces the production of reactive oxygen species and also enhances activity of the antioxidant enzyme; superoxide dismutase. It inhibits calcium oxalate crystal growth and adhesion to renal epithelial cells. Triterpenoids have been reported to have diuretic properties and reduces the risk of calcium oxalate nephrolithiasis by increasing the urinary volume, which results in reduction in the calcium oxalate supersaturation in the urine ⁶. Alkaloids are effective in prevention as well as in the treatment of calcium oxalate nephrolithiasis, exhibiting these effects through a combination of antioxidant, diuretic, hypocalciuric and urine alkalinizing activities ⁷.

Figure-1 In-vitro experimental model set-up to evaluate anti-nephrolithiatic activity

In-vitro experimental model set-up to evaluate the anti-nephrolithiatic activity is shown in fig. 1. The standard cystone (fig. 2) showed maximum dissolution of calcium stones (56%) followed by the chloroform (42%) and benzene (40%) extracts of seeds. The leaves extract of benzene showed positive results (44%) followed by chloroform (42%), ethanol (40%), methanol (40%) and ethyl acetate (38%).

Table 1: Phytochemical analysis of different extracts of M. uniflorum seeds (S) and leaves (L).

	Benzene		Chloroform		Ethanol		Ethyl acetate		Methanol
	S	L	S	L	S	L	S	L	S	L
Glycosides	-	-	+	+	+	+	-	-	+	+
Reducing Sugars	-	-	-	-	+	+	-	-	+	+
Proteins	-	-	-	-	+	+	-	-	+	-
Steroids	-	-	-	-	+	-	-	-	+	-
Triterpenoids	+	+	+	-	+	-	-	-	-	+
Flavonoids	-	-	-	-	+	+	-	-	+	+
Phenols	+	-	+	+	-	-	-	-	-	-
Alkaloids	+	+	-	-	+		+	+	+	+
Tannins	+	-	-	-	+	+	-	-	+	+
Saponins	-	-	+	+	+	+	-	-	-	-

Figure 2: Graph showing percent dissolution of calcium oxalate by different extracts of M. uniflorum leaves and seeds

The GC-MS analysis showed that the prevailing compounds in methanol extract of leaves were hexadecanoic acid, methyl ester (8.81%), 1,2,4-trioxolane-2-octanoic acid, 5-octyl-, methyl ester (8.53%),10-octadecenoic acid, methyl ester(21.04%), n-hexadecanoic acid (20.59%), oleic acid (22.10%), [1,1’-bicyclopropyl]-2-octanoic acid, 2’-hexyl-, methyl ester (12.64%), and 2-hexadecanol (6.25%) (Table 2). The spectrum profile of GC-MS confirmed the presence of seven major compounds (Fig. 3A). The individual fragmentations of the components are illustrated as (Fig. 3B-3H).

Table 2: Compounds found in GC-MS analysis of M. uniflorum leaves

S. NO	COMPOUND NAME	RT	PEAK AREA	PEAK AREA (%)
1	1,2,4-trioxolane-2-octanoic acid, 5-octyl-, methyl ester	15.72	16042608	8.53
2	hexadecanoic acid, methyl ester	17.15	16562896	8.81
3	n-hexadecanoic acid	17.87	38712432	20.59
4	10-octadecenoic acid, methyl ester	18.90	39552048	21.04
5	Oleic acid	19.65	41548544	22.10
6	[1,1’-bicyclopropyl]- 2-octanoic acid, 2’- hexyl-, methyl ester	20.08	23769200	12.64
7	2-hexadecanol	29.88	11754064	6.25

Figure 3A: GC-MS spectrum of methanol extract of M. uniflorum leaves

Figure 3B: 1, 2, 4-Trioxolane-2-octanoic acid, 5-octyl-, methyl ester (RT: 15.72)

The GC-MS analysis showed that the prevailing compounds in methanol extract of seeds were Mome-inositol (23.8%), Hexadecanoic acid (5.1%), Hexadecanoic acid, methyl ester (0.4%), Octadecanoic acid, methyl ester (0.5%), Gamma tocopherol (4.0%) (Table 3). The spectrum profile of GC-MS confirmed the presence of seven major compounds (Fig. 4A). The individual fragmentations of the components are illustrated as (Fig. 4B-4F).

Table 3: Compounds found in GC-MS analysis of M. uniflorum seeds.

S. NO	COMPOUND NAME	RT	AREA	PEAK AREA (%)
1	Mome inositol	15.19	1988651	23.8
2	Hexadecanoic acid	17.29	432190	5.1
3	Hexadecanoic acid, methyl ester	20.42	38510	0.4
4	Octadecanoic acid, methyl ester	22.79	44476	0.5
5	Gamma tocopherol	27.25	334767	4.0

Mome inositol functions as an antialopecic, anticirrhotic, antineuropathic, cholesterolytic and lipotropic. Hexadecanoic acid is an antioxidant and functions in scavenging of free radicals. Hexadecanoic methyl ester is an antioxidant, hypercholesterolemic and also functions as a lubricant. Octadecanoic acid methyl ester has anti-inflammatory, anticancer, hepatoprotective properties apart from being an antioxidant ⁸. Tocopherols have a saturated phytyl tail which is attached to their chromanol ring. Gamma tocopherol completely prevents calcium oxalate deposition, by preventing the peroxidative injury and also by restoring renal tissue antioxidants ⁹. The useful effect of vitamin E in reducing CaOx accumulation in the kidneys can be attributed to attenuation of tubular cell death and enhancement of the defensive roles of Tamm–Horsfall protein (THP). It can also preserve renal function and reduce levels of free radicals, vasoconstrictive thromboxanes and tubulointerstitial fibrosis in nephrotoxicity model in rats ¹⁰. Therefore it might provide protection against the deposition of calcium oxalate stones in kidney of humans.

4. CONCLUSION:

The in vitro results in this study showed that both seeds and leaves of M. uniflorum have anti-nephrolithiatic activity which can be validated further by in vivo studies. The presence of various compounds justifies the use of the whole plant for various diseases including kidney stones. Thus identification of bioactive compounds by GC-MS analysis is the first step towards understanding the mechanism of this medicinal plant in its anti-nephrolithiatic activity and this type of study will be helpful for further detailed study in vivo. This could be of great importance as patients would not have to undergo the expensive treatment and surgeries involved in removal of kidney stones.

5. ACKNOWLEDGEMENTS:

We are grateful to Department of Biotechnology, G.B. Pant Engineering College, Pauri, Uttarakhand and TEQIP for providing infrastructure to Amritan Kachru for carrying out research. Also we would like to thank Dr. Ajay Kumar, Scientist, AIRF, JNU, Delhi, for helping us perform GC-MS.

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Received on 07.11.2015 Modified on 26.11.2015

Res. J. Pharmacognosy & Phytochem. 8(1): Jan.- Mar. 2016; Page 05-12

DOI: 10.5958/0975-4385.2016.00002.9