IN-VITRO ANTIUROLITHIATIC POTENTIAL OF LEAVES OF EUPHORBIA HIRTA L. AGAINST CALCIUM OXALATE KIDNEY STONES
HTML Full TextIN-VITRO ANTIUROLITHIATIC POTENTIAL OF LEAVES OF EUPHORBIA HIRTA L. AGAINST CALCIUM OXALATE KIDNEY STONES
Dolly Kain, Suresh Kumar *, Amrita Suryavanshi and Atul Arya
Medicinal Plant Research Laboratory, Department of Botany, Ramjas College, University of Delhi, Delhi-110007, India.
ABSTRACT: Plant Euphorbia hirta L. is a common weed plant that belongs to the family Euphorbiaceae characterized by the presence of milky latex in leaves and stems. Phytochemical screening of leaves of Euphorbia hirta L. confirmed the presence of alkaloids, cardiac glycosides, flavonoids, phenolic compounds, tannin and terpenoids. FT-IR confirms the formation of calcium oxalate crystals with major peaks at 3331.07, 1608.63, 1317.38, and 775.38, which corresponds to asymmetric O-H bending, C=O stretching, C-O stretching and C-H bending. Nucleation and aggregation assay has been done to investigate the in-vitro antiurolithiatic potential of leaves of Euphorbia hirta L., and significant % inhibition has been observed for nucleation and aggregation of calcium oxalate kidney stones. Maximum % inhibition has been observed at 1000 µg/ml concentration with 55.81% and 73.17% inhibition, respectively. % inhibition of nucleation and aggregation increases with the increasing concentration of plant E. hirta. GC- MS confirmed the presence of different active compounds.
Keywords: |
Euphorbia hirta L., Phytochemical, Nucleation, Aggregation, calcium oxalate, FT-IR
INTRODUCTION: Urolithiasis is the third most prevailing and painful disorder of global concern. It is the process of kidney stone formation in the urinary tract. Supersaturation of the urine with crystal-forming substances and imbalance between promoters and inhibitors are two major causes of kidney stone formation. Nucleation is the first step in kidney stone formation in which the smallest unit of crystal i.e. “nuclei” or “nidus” of calcium oxalate stones formed 1, 2. When the nuclei of calcium oxalate started binding to each other and formed larger particles, a process called aggregation.
Strong intermolecular forces of crystals not allowed nuclei to get separated easily, and now these crystals are large enough to behave like a stone 1, 2. Kidney stones vary in composition and hence can be of different types like calcium oxalate, calcium phosphate, uric acid, and mixed (magnesium, ammonium, calcium, and phosphate), but calcium oxalate stones are most abundant 3. Many plants have been used in the treatment of kidney stones, and various plants are reported to have antiurolithiatic activity 4, 5. Plant Euphorbia hirta L. commonly known as dhudhi, asthma weed and dugdhika, belongs to family Euphorbiaceae with many activities reported in the literature. Present study has been designed to bring light on the urolithiatic potential of leaves of Euphorbia hirta L.
MATERIALS AND METHODS:
Collection of Plant and Preparation of Extract: Plant E. hirta has been collected from the garden of the Department of Botany, the University of Delhi in the month of July-August and identified using “Illustration to The Flora of Delhi, Page 193 and Figure 193”. Leaves of the plant were shade dried and powdered using a mixer grinder and extracted in ethanol by the soxhlet method.
Phytochemical Screening:
TABLE 1: METHODOLOGY FOR PHYTOCHEMICAL SCREENING
S. no. | Secondary metabolite | Name of test | Methodology | Observations |
1 | Alkaloids | Wagner test | 2ml extract + 1% HCl + steam + 1ml Wagner’s reagent drop by drop | Brownish red Precipitate6 |
2 | Cardiac glycosides | Kellar- Killiani test | 2ml extract + 2 ml of chloroform + H2SO4 to form a layer | Brown ring at interphase7 |
3 | Flavanoids | NaOH test | Extract + dilute NaOH, + dilute HCl | Yellow solution on NaOH turns colorless on HCl7 |
4 | Phenolic compounds | Lead acetate test | Extract + few drops of 10% lead acetate solution | Formation of white precipitate8 |
5 | Saponin | Frothing test | 0.5ml extract + 5ml distilled water and shake well | Persistence of frothing6 |
6 | Tannin | Braemer’s test | 10% alcoholic FeCl3 + 2-3ml of methanolic extract (1:1) | Dark blue or greenish grey coloration6 |
7 | Terpenoids | Salkowski test | 5ml extract + 2ml Chloroform + 3ml conc. H2SO4 | Reddish Brown color of interface6 |
8 | Anthroquinone | Ammonia test | 1 ml dilute (10 %) ammonia + 2ml extract | A pink-red color in the ammoniacal (lower) layer7 |
9 | Phlobatannin | HCl test | Extract boiled with 2 ml of 1% hydrochloric acid | Formation of red precipitate8 |
10 | Starch | Iodine test | 2ml extract + 2ml iodine solution | Formation of Blue color9 |
Nucleation Assay: The method used was similar to that described by Hennequin et al., 10 with some minor modifications. Solutions of calcium chloride and sodium oxalate were prepared at a final concentration of 3 mmol/L and 0.5 mmol/L, respectively, in a buffer containing Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 5.5.1.9 ml of calcium chloride solution was mixed with 200 µL of the herb extract at different concentrations and incubated for 30 min at 37 ºC in a water bath. Crystallization was started by adding 1.9 ml of sodium oxalate solution. The OD of the solution was monitored at 620 nm for 420 sec.
% Inhibition = {(Abs. Control - Abs. Sample) / Abs. Control} × 100
Aggregation Assay: The method used was similar to that described by Hess et al., 11 with some minor modifications. `Seed' CaOx monohydrate (COM) crystals were prepared by mixing calcium chloride and sodium oxalate at 50mmol/L. Both solutions were equilibrated to 60 ºC in a water bath for 1 h and then cooled to 37 ºC overnight. The crystals were harvested by centrifugation and then evaporated at 37 ºC. COM crystals were used at a final concentration of 0.8mg/mL, buffered with Tris 0.05mol/L and NaCl 0.15mol/L at pH 5.7. 1 ml extract was taken in a test tube to which 3 ml COM crystal solution was added and incubated 37֯ ºC, and readings were recorded at a different time interval of 30, 60, 90 and 120 min.
% Inhibition = {(Slope Control - Slope Sample) / Slope Control} × 100
RESULTS AND DISCUSSION:
Phytochemical Screening: Phytochemical screening of leaves of Euphorbia hirta L. confirms the presence of different phytochemical groups Table 2.
TABLE 2: PHYTOCHEMICAL SCREENING
S. no. | Secondary metabolite | Name of test | Results |
1 | Alkaloids | Wagner test | + |
2 | Cardiac glycosides | Kellar- Killiani test | + |
3 | Flavanoids | NaOH test | + |
4 | Phenolic compounds | Lead acetate test | + |
5 | Saponin | Frothing test | - |
6 | Tannin | Braemer’s test | + |
7 | Terpenoids | Salkowski test | + |
8 | Anthroquinone | Ammonia test | - |
9 | Phlobatannin | HCl test | - |
10 | Starch | Iodine test | - |
Nucleation Assay and Aggregation Assay: Plant Euphorbia hirta L. showed significant % inhibition against nucleation of calcium oxalate stones with a maximum of 55.81% inhibition at 1000 µg/ml concentration Fig. 1A. Plant Euphorbia hirta L. showed significant % inhibition for aggregation of calcium oxalate nidus or crystals to form stone-like structure with maximum 73.17% inhibition at 1000 µg/ml concentration Fig. 1B. Due to nucleation of calcium oxalate nuclei, absorption at 620 nm increases but gradually absorption starts falling down due to aggregation of these nuclei to form stones 10, as observed with the leaves of Euphorbia hirta L. Fig. 1C.
FT-IR spectra Fig. 2 confirms the formation of calcium oxalate crystals as per aggregation assay protocol as it shows major peaks at 3331.07, 1608.63, 1317.38 and 775.38 which corresponds to asymmetric O-H bending, C=O stretching, C-O stretching and C-H bending 13. In aggregation assay maximum negative slope recorded at 1000 µg/ml concentration with equation y = -0.011x + 0.434 (R²=0.993) and y=-0.041x+0.359 (R²=0.982) for control. y=-0.013x+0.407(R²=0.993), y=-0.024x + 0.350(R²=0.992), y=-0.032x+0.317(R²=0.994) and y=-0.033x+0.316(R² = 0.993) at 750, 500, 250, 100 µg/ml concentration respectively.
FIG. 1: NUCLEATION ASSAY (A) AND AGGREGATION ASSAY (B AND C) OF PLANT EUPHORBIA HIRTA L.
FIG. 2: FT-IR SPECTRA OF CALCIUM OXALATE CRYSTALS
Aryal et al., 14 studied 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. 55.81% inhibition for nucleation is the significant figure as many plants like Hirniaria hirsuta favors the nucleation of crystals having a significant inhibitory effect for aggregation 15. Antiurolithiatic potential of plant E. hirta has been studied by Pauzi et al., with 95.78%, 76.38%, 38.95%, and 28.42% inhibition for aggregation of calcium oxalate stones of hexane extract, water extract, ethyl acetate extract, and methanol extract, respectively 16, which is comparable to our study with maximum % inhibition of 73.17%.
CONCLUSION: Plant E. hirta shows significant antiurolithiatic potential against calcium oxalate kidney stones and hence can be considered as a better herbal alternative for the treatment of kidney stones. It can be further studied by in-vivo methods for more clarity as per its clinical significance and also its role as an herbal drug in human health and medicine.
ACKNOWLEDGEMENT: We would like to thank the UGC (University Grant Commission) for providing fellowship to conduct research work. Our sincere thanks to Dr. Manoj K. Khanna, Principal, Ramjas College, the University of Delhi, for the necessary facilities, and we are also thankful to Prof. K.S Rao and Prof. Veena Agarawal, Department of Botany, the University of Delhi for their support and valuable suggestions.
CONFLICTS OF INTEREST: Nil
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How to cite this article:
Kain D, Kumar S, Suryavanshi A and Arya A: In-vitro antiurolithiatic potential of leaves of Euphorbia hirta L. against calcium oxalate kidney stones. Int J Pharm Sci & Res 2021; 12(2): 991-94. doi: 10.13040/IJPSR.0975-8232.12(2).991-94.
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Article Information
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991-994
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English
IJPSR
D. Kain, S. Kumar *, A. Suryavanshi and A. Arya
Medicinal Plant Research Laboratory, Department of Botany, Ramjas College, University of Delhi, Delhi, India.
suresh.kumar@ramjas.du.ac.in
06 February 2020
26 June 2020
30 June 2020
10.13040/IJPSR.0975-8232.12(2).991-94
01 February 2021