INTRODUCTION:

Urolithiasis is a complex process, which results in the formation of kidney stones and includes three important steps i.e. super saturation of urine, nucleation, and aggregation. Kidney stones vary in composition but most common are calcium oxalate stones. Various plants have been successfully used in the treatment of kidney stones including Tribulus terrestris¹, Asparagus racemosus², Pergularia daemia³, Ipomoea eriocarpa⁴, Bryophyllum pinnatum⁵ and Aerva lanata⁶. Ethnobotanical surveys of India reveal the potential medicinal plants used by local healers including antiurolithiatic potential^7,8 which, may prove as an effective alternative in the treatment of urolithiasis.

Ethnobotanical surveys of Manipur, India reveal the medicinal properties of Anneslea fragrans Wall. and its use by local healers⁹. A. fragrans is a shrub or small tree having height of 3- 15m. Leaves are highly leathery of pale green or glaucescent green color with axillary pale-yellow flowers in a corymb. Several varieties of A. fragrans have recognized primarily on leaf character differences. The current study has designed to evaluate the in- vitro antiurolithiatic potential of leaves of A. fragrans at different stages of calcium oxalate stone formation using three methods i.e. turbidity changes in artificial urine method, nucleation assay, and aggregation assay.

MATERIALS AND METHODS:

1. Collection, identification, and preparation of extract of Anneslea fragrans Wall.:

A. fragrans has collected from Manipur, India and identified by “Flora of Manipur” by N.P. Singh, A.S. Chauhan and M.S. Mondal. Leaves of plant were shade dried and grinded using mixer- grinder for extraction. Extract of leaves has prepared in methanol using soxhlet method for 48 hrs.

2. Chemicals used:

All chemicals (sodium chloride, sodium phosphate, sodium citrate, magnesium sulfate, sodium sulfate, potassium chloride, calcium chloride, sodium oxalate, ammonium hydroxide, and ammonium chloride, Tris and NaCl) that were used are of good quality and purchased from Fisher Scientific International, Inc.

3. Turbidity changes in artificial urine:

The artificial urine (AU) was prepared according to the method of Burns and Finlayson¹⁰ with the following composition- sodium chloride 105.5mmol/1, sodium phosphate 32.3mmol/1, sodium citrate 3.21mmol/l, magnesium sulfate 3.85mmol/1, sodium sulfate 16.95 mmol/1, potassium chloride 63.7mmol/1, calcium chloride 4.5mmol/1, sodium oxalate 0.32mmol/1, ammonium hydroxide 17.9mmol/1, and ammonium chloride 0.0028mmol/1. The AU was prepared fresh each day and pH adjusted to 6.0.

Study without inhibitor: A volume of 2ml of AU transferred into the cell and 1ml of distilled water added to it and blank reading taken. 1ml of 0.01 M sodium oxalate added, to the previous volume, and the measurement immediately started for a period of 420 sec¹¹.

Study with inhibitor: Extract resuspended in distilled water, filtered, and used at a final concentration of 100, 250, 500, 750 and 1000µg/ml. A mixture of 2ml of AU and 1ml of plant extract solution is versed in the cell. A blank reading has taken and then volume of 1ml of 0.01M sodium oxalate has added and the measurement is immediately started for a period of 420 sec¹¹.

% Inhibition = {(Abs. Control- Abs. Sample)/ Abs. Control} * 100

4. Nucleation assay:

The method used was similar to that described by Hennequin et al.¹² with some minor modifications. Solutions of calcium chloride and sodium oxalate were prepared at a final concentration of 3mmol/L and 0.5 mmol/L, respectively, in a buffer containing Tris 0.05mol/l and NaCl 0.15mol/l at pH 5.5. 1.9ml of calcium chloride solution mixed with 200µl of the herb extract at different concentrations and incubated for 30 minutes at 37ºC in water bath. Crystallization started by adding 1.9 ml of sodium oxalate solution. The OD of the solution monitored at 620nm for 420 s.

% Inhibition = {(Abs. Control- Abs. Sample)/ Abs. Control} * 100

5. Aggregation assay:

The method used was similar to that described by Hess et al.¹³ with some minor modifications. `Seed' CaOx monohydrate (COM) crystals were prepared by mixing calcium chloride and sodium oxalate at 50mmol/L. Both solutions equilibrated to 60ºC in a water bath for 1h and then cooled to 37ºC overnight. The crystals harvested by centrifugation and then evaporated at 3 C. COM crystals used at a final concentration of 0.8mg/ml, buffered with Tris 0.05mol/l and NaCl 0.15mol/l at pH 5.7. 1ml extract was taken in test tube to which 3 ml COM crystal solution was added and incubated 37ºC and reading were recorded at different time interval of 30, 60, 90, and 120 min.

% Inhibition = {(Slope Control- Slope Sample)/ Slope Control} *100

6. FT- IR analysis:

Fourier transform- infrared (FT- IR) spectroscopy of leaf extract of A. fragrans has performed by using Brukers spectrometer in the range of 1000cm^-1- 3500 cm^-1.

7. Statistical analysis:

Performed by calculating±SEM and strong regression (r²) value using Microsoft excel 2007

RESULTS:

1. In- vitro antiurolithiatic activity of leaves of Anneslea fragrans Wall.:

A. fragrans has shown significant in- vitro antiurolithiatic potential with 78.1%, 32.32%, and 57.11 % inhibition at 1000µg/ml concentration for turbidity changes in artificial urine assay, nucleation assay and aggregation assay respectively (Figure 1(A), 1(B), and 1(C)).

2. Fourier transform- infrared (FT- IR) analysis of leaves of Anneslea fragrans Wall.:

FT-IR analysis reveals different peaks corresponding to major functional groups including hydroxyl, saturated aliphatic, quinone or conjugated ketone and aliphatic flouro group (Figure 2 and Table 1).

Table 1: FT-IR analysis of leaves of A. fragrans

Peak value (cm^-1)	Type of stretching vibration	Functional group
3307.84	O-H stretching	Hydroxyl group
2927.26	Methylene C-H asym./sym. stretching	Saturated aliphatic group (alkane/alkyl)
1610.37	C-H stretching	Quinone or conjugated ketone
1446.19	Methyl CH asym./sym. bending	Saturated aliphatic group (alkane/alkyl)
1044.02	C-F stretching	Aliphatic Flouro compounds

DISCUSSIONS:

In- vitro antiurolithiatic potential of different plant samples have evaluated by different scientists including Vamsi et al., 2014¹⁴ have studied the antiurolithiatic potential of of Mucuna pruiens through turbidity changes in artificial urine method with 63.1% inhibition at 250µg/ml concentration whereas Vennila et al., 2015¹⁵ reported 98% inhibition in Melia dubia leaves at 100mg/ml concentration. Similar assay of nucleation and aggregation has also been performed by Atmani and Khan, 2000¹⁶ on Herniaria hirsuta, by Binu and Vijayakumari, 2016¹⁷ on Strychnos potatorum and by Chandirika and Annadurai, 2018¹⁸ on Lantana camara. Aryal et al., 2019¹⁹studied nucleation and aggregation activity of Achyranthes aspera, Lawsonia inermis, Ficus benghalensis, Raphnus sativus and Macrotyloma uniflorum and found maximum nucleation and aggregation activity in R. sativus i.e. 55.21% and 61.6% inhibition respectively. FT- IR analysis has been used to characterize the different plant extract including Ajuga parviflora Benth leaf extract²⁰, Achillea millefolium L. different extracts²¹, chitosan nanoparticles of Achillea millefolium L. inflorescence extract²². Antiuolithiatic activity of different plant extract has dertermined by using ethylene glycol induced rats such as Betula utilis²³, Plectranthus tomentora²⁴, Macrotyloma uniflorum²⁵, Terminalia arjuna^26.Similar in- vitro antiurolithiatic activity of different plant extract has also been determined in the literature including Terminalia arjuna²⁶, Costus igneus²⁷, Piper nigrum²⁸, polyherbal formulation (lithout tablets)²⁹, Vigna radiata³⁰, comparision of Aerva lanata, Sphaeranthus indicus and Merremia emarginata³¹, Macrotyloma uniflorum³². Anneslea fragrans Wall. has shown excellent in- vitro antiurolithiatic activity and hence, can be consider as an effective herbal alternative or constituent of herbal formulation for treatment of urolithiasis. Further study can be done to reveal the in- vivo potential of A. fragrans against calcium oxalate kidney stones.

ACKNOWLEDGEMENT:

The author is thankful to the Principal, Dr. Manoj K. Khanna, Ramjas College, University of Delhi, Delhi for providing necessary facilities and encouragement during the course of investigation. Author is also thankful to SERB, DST, GOI for financial support.

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Received on 15.12.2020 Modified on 19.07.2021

Research J. Pharm.and Tech 2022; 15(4):1671-1674.

DOI: 10.52711/0974-360X.2022.00279