Comparison of In vitro Antiurolithiatic Activity of Aerva lanata, Sphaeranthus indicus, Merremia emarginata

 

Neeraja Kamakshi. U*¹, Ganga Rao. B², Venkateswara Rao. B³

¹Dept. of Pharmacognosy, K.C. Reddy Institute of Pharmaceutical Sciences, Jangamguntlapalem, Guntur, Andhra Pradesh, India.

²Dept. of Pharmacognosy, A.U College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India.

³Dept. of Pharmaceutical Analysis, Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Chowdavaram, Guntur, Andhra Pradesh, India.

 

ABSTRACT:

Urolithiasis is a term used to describe calculi or stones formed in urinary tract. It involves the formation of calcifications in the urinary system, usually in the kidneys or ureters, but may also affect the bladder or urethra. It is a serious, debilitating problem in all societies throughout the world, affecting approximately, 12% of the population and men are three times more prone than women. It is more prevalent between the ages of 20 and 40 in both sexes. Present study is one of the attempts to compare Antilithiatic activity of various popularly known herbs with marketed formulation Cystone. Methanolic extracts of Aerva lanata, Sphaeranthus indicus, Merremia emarginata were compared with standard drug respectively by Spectrophotometric estimation of Calcium Oxalate and Calcium Phosphate dissolution models. Homogenous precipitation method was used to prepare artificial stones such as Calcium Oxalate and Calcium Phosphate and semi-permeable membrane of eggs was used as dissolution bags. Dissolution models were incubated for 72hrs and after that, the entire content in dissolution bags was estimated spectrophotometrically at 620 nms. In dissolution models all the extracts exhibited better dissolution of Calcium Oxalate and Calcium Phosphate crystals. However Cystone exhibited strong inhibitory action, whereas Methanolic extract of Aerva lanata showed almost similar activity, remaining shows considerably less inhibitory action at different concentrations than Standard. Phytochemical analysis revealed that all the plants showed presence of Alkaloids, Glycosides, Flavanoids, Lipids. Finally concluded that one of the above constituent may be responsible for the present activity. Further evaluation of these extracts as formulations are to be carry out.

 

 

 

INTRODUCTION:

Calculi in the urinary system are called urinary calculi and include kidney stones (renal calculi or nephroliths) and bladder stones (vesical calculi or cystoliths)^[1].

 

Urolithiasis is a complex process that occurs from series of several physicochemical events like super-saturation, nucleation within kidneys. Calcium-containing stones, especially Calcium Oxalate, basic Calcium Phosphate, Non calcareous stones (non radio-opaque) Struvite (Magnesium ammonium phosphate), Uric acid (UA), Cystine , over use of synthetic drugs which results in higher incidence of adverse drug reactions ^[2]. Kidney stones are a common cause of blood in the urine and pain in the abdomen, flank, or groin. The size of kidney stones ranges from tiny microscopic crystals to stones as large as potatoes. Studies suggest that the initial factor involved in the formation of a stone may be the presence of Nano bacteria that form a calcium phosphate shell ^[3, 4]. Many stones are asymptomatic and discovered during investigations for other conditions. The classical features of renal colic are sudden severe pain. It is usually caused by stones in the kidney, renal pelvis or ureter, causing dilatation, stretching and spasm of the ureter. Renal stones are common, being present at some time in one in ten of the population, although a significant proportion will remain asymptomatic^[5]. Complications from kidney stones are uncommon (although the pain at the time can be severe). Sometimes a large stone can completely block the passage of urine down one of the tubes draining urine from the kidney (the ureter). This may lead to infection or damage to the kidney. Kidney stone can be prevented from developing again by having plenty to drink, by avoiding oxalate rich diet like coffee, Spinach; regular intake of medicine^[6]. Sphaeranthus indicus (Asteraceae)^[11] is a genus of India, Sri Lanka, Africa and Australia. According to Ayurveda, this herb is laxative, digestible, tonic, anthelmintic and nephroprotective. Merremia emarginata (Convolvulaceae)^[11] is a genus of flowering plants which is prevalent throughout India, Malaysia and tropical Africa. It is an uncultivated food crop used as a green leaf vegetable by poor people in India .The plant is traditionally used as diuretic and for cough, headache, neuralgia, rheumatism, inflammation, troubles of nose and fever due to enlargement of the liver and also for treating cancer ^[7]. Euphorbia hirta (Euphorbiaceae)^[11] is native to India but is a pan tropical weed, found on wasteland. It is a small, erect or ascending annual herb with hairy stems. The plant has been used for female disorders but is now more important in treating respiratory ailments and as diuretic ^[8].Milk of the plant is using to treat various eye related ailments.

 

MATERIALS AND METHODS:

Chemicals:

All the chemicals use for the experiment was analytical grade and were procured from National Scientifics Ltd, Guntur, Andhra Pradesh. Cystone® was purchased from Himalaya Drug Company.

Collection of Plant:

All the Plant materials were collected from Rice crop fields of Narakoduru village, Guntur District, Andhra Pradesh and were authenticated by Dr. M. Raghu Ram, Assistant professor of Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh.

 

Extraction:

All the plants were washed thoroughly with water, dried and powdered. All the powder was extracted with methanol by using Soxhlet extractor. All the extracts were poured individually to a pre-weighed china dish and concentrated to dryness over water bath and the dried residue was made moisture free in a desiccators. The extract thus prepared was used for phytochemical tests, In-vitro Antiurolithiatic activity by homogenous precipitation method.

 

Phytochemical Screening:

Methanolic extracts of these plants were screened qualitatively for the presence of various phytochemical constituents by standard procedure ^[12-15].

 

In-vitro Antiurolithiatic activity ^[9]:

Experimentally kidney stones were prepared by homogenous precipitation method. Semi - permeable membrane was removed chemically by placing the eggs in 2M HCl for overnight, which caused complete decalcification. Then the membranes were washed thoroughly with distilled water, stored in refrigerator at a pH of 7-7.4.

 

Preparation of Standard Solution:

A poly herbal formulation such as Cystone was selected and tablets were placed in absolute ethanol for removing colour coating and were crushed into powder form.  The powder was dispersed into 100ml of distilled water and filtered. Filtrate was used as positive control.

 

Estimation of CaOx and CaPo₄ by Titrimetry ^[10]:

Comparison of Antilithiatic activity by using standard Cystone tablets were carried out by taking control, Standard, Test groups.

 

 

Table 1: In-vitro Antilithiatic activity of CaOx Dissolution model

Control	Standard	Test
		Aerva lanata	Sphaeranthus indicus	Merremia emarginata
1ml (1mg/1ml) CaOx +1ml water	1ml CaOx +1ml (400 mg/ml) Cystone	1ml CaOx +1ml (100mg/ml) ALME	1ml CaOx +1ml (100mg/ml) SIME	1ml CaOx +1ml (100mg/ml)  MEME
		1ml CaOx +1ml (200mg/ml) ALME	1ml CaOx +1ml (200mg/ml) SIME	1ml CaOx +1ml (200mg/ml)  MEME
		1ml CaOx +1ml (300mg/ml) ALME	1ml CaOx +1ml (300mg/ml) SIME	1ml CaOx +1ml (300mg/ml) MEME
		1ml CaOx +1ml (400mg/ml) ALME	1ml CaOx +1ml (400mg/ml) SIME	1ml CaOx +1ml (400mg/ml) MEME

ALME: Aerva lanata Methanolic extract, SIME: Sphaeranthus indicus Methanolic extract, MEME: Merremia emarginata Methanolic extract, CaOx:

Calcium Oxalate:

All the models were prepared by packing in semi permeable membrane as mentioned above. The membranes were sutured and were allowed to suspend in 100ml of 0.1M Tris buffer. All the flasks were subjected to incubation, preheated to 37⁰C for 7hrs for 3days. After 3 days content of each membrane was collected in different test tubes. 2ml of 1N Sulphuric acid was added to each test tube and titrated with 0.9494N KMnO4 till the colour disappears. The amount of undissolved Calcium oxalate is subtracted from the total quantity used in the experiment in the beginning; to know much quantity of Calcium oxalate actually test substances could dissolve.

 

Table 2: In-vitro Antilithiatic activity of CaPo₄ Dissolution models

Control	Standard	Test
		Aerva lanata	Sphaeranthus indicus	Merremia emarginata
1ml (1mg/1ml) CaPo4 +1ml  water	1ml CaPo4 +1ml (400 mg/ml)  Cystone	1ml CaPo4 +1ml  ALME 100mg/ml	1ml  CaPo4 +1ml SIME 100mg/ml	1ml  CaPo4 +1ml  MEME 100mg/ml
		1ml  CaPo4 +1ml ALME 00mg/ml	1ml CaPo4 +1ml SIME 200mg/ml	1ml CaPo4 +1ml  MEME 200mg/ml
		1ml  CaPo4 +1ml  ALME 300mg/ml	1ml  CaPo4 +1ml SIME 300mg/ml	1ml CaPo4 +1ml  MEME 300mg/ml
		1ml  CaPo4 +1ml ALME 400mg/ml	1ml CaPo4 +1ml SIME 400mg/ml	1ml CaPo4 +1ml MEME 400mg/ml

 

 

All the models were prepared by packing in semi permeable membrane as mentioned above. The membranes were sutured and were allowed to suspend in 100ml of 0.1M Tris buffer. All the flasks were subjected to incubation, preheated to 37⁰C for 7hrs for 3days. 4ml of 1N H2SO4 and 3ml of molybdate-sulphuric acid reagent, 1ml of reducing solution were added and kept aside for 2hrs. Change in colour intensity was measured against 620nm spectrophotometrically. The amount of un dissolved Calcium  Phosphate is subtracted from the total quantity used in the experiment in the beginning, to know  much quantity of Calcium  phosphate actually test substances could dissolve.

 

                   (weight of precipitate  in blank set)-

                                      (weight of precipitate in experimental set)

% Inhibition =     ------------------------------------------- X 100

                                (weight of precipitate  in blank set)

 

RESULTS AND DISCUSSION:

Preliminary phytochemical screening for Methanolic extract of Aerva lanata revealed the presence of carbohydrates/glycosides, phenols, steroids, tannins. Alkaloids, β-Sitosterols, isoflavone, terpenoids were identified in Methanolic extract of Sphaeranthus indicus. Merremia emarginata have shown the presence of Carbohydrates, Flavonoids, Alkaloids, and Lipids. The phyto chemical constituents present in the extract can be held responsible for different medicinal activities of the plant.  Cystone a prescribed medicine for renal calculi showed highest inhibition of both CaOx and CaPo₄ mineralization. ALME showed almost similar inhibition whereas SIME, MEME showed considerably less inhibition when compared with the standard Cystone at different concentrations.

 

 

Figure1: Comparison of % inhibition of CaOx at different concentrations

Table3: Percentage inhibition of CaOx mineralisation in dissolution models

 

Table4: Percentage inhibition of CaPo₄ mineralisation in dissolution models

 

Figure2: Comparison of % inhibition of CaPo4 at different concentrations

 

 

CONCLUSION:

From the above results it was concluded that all the plant species individually having significant Antiurolithiatic activity. The plant extracts reveals the presence of carbohydrates, steroids, tannins, phenolic compounds, flavonoids and terpenoids. When compared with standard Cystone all the extracts showed considerable Antiurolithiatic activity. Further characterization of its active constituents to be done. Methanolic extracts of all the species could be further evaluated In vivo.

 

ACKNOWLEDGEMENT:

The authors are thankful to the management of K C Reddy Institute of Pharmaceutical Sciences, Jangamguntlapalem, and Guntur for providing the facilities and constant support to carry out the research work.

 

REFERENCES:

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12.     Kokate CK. Text book of Pharmacognosy. 16^th Edition, Nirali Prakhasan Publishers, Mumbai, India, 2001.

13.     Manoj Goyal et al. Aerva lanata: A review on Phytochemistry and Pharmacological aspects. Pharmacognosy Review. 2011; 5(10):195–198.

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15.     Rajagopal PL, Robin Jose, Sreejith KR, Kiron SS, Premaletha K, Aneeshia S. Phytochemical and Anthelmintic studies on the whole plant of Merremia emarginata Nurm. F. American Journal of Pharmacy and Health Research. 2004; 2(7):72-76.

 

 

 

Accepted on 27.04.2017        © RJPT All right reserved