DESC:Field of the invention
This invention relates to a novel herbal composition of aqueous extract of Capparis decidua and its antinephrolithiasis activity.
Background of the invention
Nephrolithiasis
A kidney stone is like a small rock that forms in the kidney. Stones form when certain chemicals in the body clump together. A stone can either stay in the kidney or travel through the urinary system. Anyone can have a kidney stone, but it may be more likely if you:
• Are male
• Are Caucasian
• Are very overweight
• Have had kidney infections
• Have a family member with kidney stones
• Have had kidney stones before
• Eat a lot of animal protein (such as meat and eggs)
• Do not drink enough liquids

Other conditions and medicines can also put you at greater risk for kidney stones.
Very small stones might pass through the urinary system without causing much pain. Larger stones can block the flow of urine if they get stuck in the ureters or urethra. Kidney stones do not usually cause any symptoms until they start to pass. Some symptoms might include:
• Extreme pain in your back or side that will not go away
• Throwing up
• Blood in your urine
• Fever and chills
Sushila Rathee et. al.(2010) Capparis decidua (Forsk.) is belonging to family Capparidaceae, yet important medicinal plant of Indian Medicinal Plants. In the traditional system of medicine, the bark has been shown to be useful in the treatment of coughs, asthma and inflammation; roots used in fever and buds in the treatment of boils. In Unani, leaves act as appetizer, helps in cardiac troubles, fruits used in biliousness.
The plant is reported to contain Phytochemicals including alkaloids, terpenoids, glycosides and some fatty acids. In Unani, leaves act as appetizer, helps in cardiac troubles, used in biliousness; alveolaris and pyorrhea; Root bark is used as anthelmintic and purgative. The plant have significant pharmacological activities like hypercholesterolemic, anti-inflammatory and analgesic, antidiabetic, antimicrobial, antiplaque, antihypertensive, antihelmintic & purgative activities. The review analyses phytochemical and pharmacological potential of medicinal plant (kair).
Subrata Kumar Biswas et.al.(2011) found that this plant showed various pharmacological activities such as anthelmentic, immunosuppressive, wound healing, hepatoprotective, antinociceptive, anti-inflammatory and antidiabetic, nephroprotective, antioxidant activity, antimicrobial activity, analgesic, anticonvulsant, neuropharmacological and antipyretic.
Anthelmentic activity was found due to the presence of tannins of the extract of K. pinnata and steroid glycosides such as bufadienolide showed wound healing activity.
It was also found that the different flavonoids, polyphenols, triterpenoids and other chemical constituents of the plant were responsible for the antinociceptive, anti-inflammatory and antidiabetic properties. Quercetin had a marked protective effect on cadmium induced nephrotoxicity and possessed potent oral efficacy against cutaneous leishmaniasis.
Patel Sanjay et. al., 2009 studied the anticancer activity of the fruits of Solanum nigrum on the HeLa cell lines. The fruits of Solanum nigrum methanolic extract were tested for its inhibitory effect on HeLa cell line. The percentage viability of the cell line was carried out by using Trypan blue dye exclusion method. The cytotoxicity of Solanum nigrum on HeLa cell was evaluated by the SRB assay and MTT assay.
Shukla Sangita et. al., 2009 assess diuretic potential of methanol and aqueous extracts of the aerial parts was assessed in albino rats using in-vivo Lipschitz test model. The volumes of urine, urinary concentration of sodium and potassium ions were the parameters of the study. Frusemide was used as standard. The results indicate that methanol and aqueous extract at 500 mg/kg body weight shows a significant (p<0.05) increase in the urine volume and electrolyte excretion (p<0.001) when compared to control. Both the extracts show significant diuretic activity.
Seema V. Pattewar et.al. 2012 provide advance information for the drug discovery research from the divine herb Kalanchoe pinnata, which contains a wide range of active compounds, including alkaloids, triterpenes, glycosides, flavonoids, steroids, bufadienolides, lipids and organic acids. The pharmacological studies are reviewed and discussed, focussing on activities as immunomodulator, CNS depressant, analgesic, antimicrobial, antiinflammatory, antiallergic, antianaphylactic, antileishmanial, antitumorous, antiulcerous, antibacterial, antifungal, antihistamine, antiviral, febrifuge, gastroprotective, immunosuppressive, insecticidal, muscle relaxant, sedative, anticancer.
Lieske John C.et.al 2000 The binding and internalization of calcium oxalate monohydrate (COM) crystals by tubular epithelial cells may be a critical step leading to kidney stone formation. Exposure of MDCK cells to arachidonic acid (AA) for 3 days, but not oleic or linoleic acid, decreased COM crystal adhesion by 55%. Exogenous prostaglandin PGE1 or PGE2 decreased crystal binding 96% within 8 h, as did other agents that raise intracellular cAMP. Actinomycin D, cycloheximide, or tunicamycin each blocked the action of PGE2, suggesting that gene transcription, protein synthesis, and N-glycosylation were required.
Blockade of crystal binding by AA was not prevented by the cyclooxygenase inhibitor flurbiprofen, and was mimicked by the nonmetabolizable AA analog eicosatetryanoic acid (ETYA), suggesting that generation of PGE from AA is not the pathway by which AA exerts its effect. These studies provide new evidence that binding of COM crystals to renal cells is regulated by physiological signals that could modify exposure of cell surface molecules to which the crystals bind. Intrarenal AA, PGs, and/or other agents that raise the intracellular concentration of cAMP may serve a protective function by preventing crystal adhesion along the nephron, thereby defending the kidney against crystal retention and stone formation.

Yung-Ching Chien et.al.(2009) Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin / uropontin (OPN) are commonly found in kidney stones. Crystals grown with OPN showed increased dimensions of the prismatic faces attributable to selective inhibition at this crystallographic face.
At high concentrations of OPN, elongated crystals with dominant faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp86–93 dose-dependently elongated crystal morphology along the faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp86–93 followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into crystal sectors. Computational modeling of poly-Asp86–93 adsorption to COD and surfaces also suggests increased stabilization of the COD surface and negligible change to the natively stable surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN.

SUMMARY OF THE INVENTION
A kidney stone is like a small rock that forms in the kidney. Stones form when certain chemicals in the body clump together. A stone can either stay in the kidney or travel through the urinary system. Very small stones might pass through the urinary system without causing much pain. Larger stones can block the flow of urine if they get stuck in the ureters or urethra. Kidney stones do not usually cause any symptoms until they start to pass. The study was undertaken to investigate the anti nephrolithiasis activity of Capparis decidua. In preventive regimen AECD 800 mg/kg (57.35% relief) show greater Creatinine clearance then standard drug cystone (34.54% relief) and in curative regimen cystone (125.33% relief) has greater effect then AECD 800 mg/kg (42.48% relief). Test drug show dose dependent therapeutic efficacy. Therapeutic effect of test drug increase with increasing dose.
The present invention can be claimed in following manner:
1. A method of making of pharmaceutical composition of Aqueous extract of Capparis decidua (AECD) comprising the steps of:
collecting Capparis desidua from it origin and drying to powered form;
mixing the powdered form with a sufficient volume of distilled water and extracted with a soxhlet apparatus in a solvent;
Removing the solvent and extract is dried in an oven and weighted.

2.The method as claimed in claim 1, wherein said powdered form with a sufficient volume of distilled water and extracted with a soxhlet apparatus for 16 to 18 hours.
3. The method as claimed in claim 1, wherein said extract is dried in an oven with the temperature of 50°-60°C.
4. A pharmaceutical preparation of Aqueous extract of Capparis decidua (AECD) comprises 800 mg/kg (4.76 ml/min, 57.35% relief) show greater Creatinine clearence then standard drug cystone (4.07 ml/min, 34.54% relief) and in curative regimen cystone (5.14 ml/min, 125.33% relief) has greater effect then AECD800 mg/kg (3.25 ml/min, 42.48% relief).
5. The pharmaceutical preparation of Aqueous extract of Capparis decidua (AECD) as claimed in claim 4, wherein said preparation is used in antinephrolithiasis activity.

Brief Description of the Drawings
Fig 1: Photograph of Capparis deciduas
Fig 2: Anti nephrolithiatic Activity (Preventive regimen)
Fig 3: Anti nephrolithiatic Activity (Curative regimen)
DETAILED DESCRIPTION OF THE DRAWINGS
ACTIVITY DESCRIPTION –
Plant profile Capparis desidua:
Collection and Identification of Plant Material
The chosen plant, Capparis decidua was identified and selected by the experts of Botany Department, Institute of biomedical and industrial research, Jaipur. For the present study various parts of the Capparis decidua i.e., fruits, flowers and barks were collected in and around Jaipur (India).
It is commonly known as karer, karel, kenr, karu etc is a densely branching shrub or small tree of the Thar Desert.
It is also found in the subtropical and tropical zones and other arid regions in southern Asia with a mass of slender, 4-5 meter high or occasionally a small tree with many green vines like apparently leafless branches, hanging in bundles. The bark turns into whitish- grey colour with age but most of the branches and twigs are a glossy dark green in colour. Small light brown spines occur in pairs on the twigs at each mode. Leaves are very minute (2 mm long) with a very short life span on young shoots, so that the plant looks leafless most of the time. The new flush of leaves appears in November – January. Flowers are pink in colour, red veined in small groups along the leafless shoots, in the axils of spines. Fruits are small many seeded ovoid or sub-globulous, slightly mucronate pink berry of the size and shape of a cherry, becomes blackish when dry. (Sushila Rathee et.al)
Phytoconstituent-
The plant possesses a number of alkaloids, terpenoids, glycosides and some fatty acids. The different phytoconstituents of different plant parts are as follow-Capparine, Cappariline and capparinine The bark shows the presence of n-pentacosane, n-tricontanol and ß-sitaosterol besides a water-soluble alkaloid, 1-stachydrine .
Besides these, six new phytoconstituents have been isolated and characterized from the root bark, which are capparisterol, Capparideciduasterol, Capparisditerpenol, in aliphatic hydroxyketone and capparisditerpenyl ester.(deepak et. al)
Material and Method–
Equipment –
Soxhlet assembly, Semi auto analyzer, Hot air oven, Water Bottles, Polypropylene rat Cages etc.
Chemical and Drugs –
Ethylene glycol, Cystone, Sodium azide. Diethyl ether, Formaldehyde, Ethanol, and Test Kit For Urinary LDL, Serum Urea, Serum Creatinine, Crcl etc.
Animals –
Albino Wistar Rat (150-175gm)
Prepration of extract of Capparis decidua
Capparis desidua was collected from the campus of Garden of Department of dravyaguna vigyan, National Institute of Ayurveda. Whole plant was dried and powdered. Then 100 g of the powder was mixed with a sufficient volume of distilled water and extracted with a soxhlet apparatus for 16 to 18 hours. The solvent was removed and the extract was dried in an oven with the temperature of 50°-60°C and weighted.

Toxicity studies according to OECD Guideline 420
The toxic dose was determined on the basis of a pre-studied experiment which was carried out on the rats. Four doses that were less than the lethal dose (LD50) (2000 mg/kg, 4000 mg/kg, 6000 mg/kg and 8000 mg/ kg as lower and higher dose, respectively) were taken as effective dose in the current study.

Four doses for aqueous extract for Capparis decidua extract to achieve lethal dose (LD50) were taken as effective dose in the current study.
Groups are divided as follow:
Group A: 2000 mg/kg; 10 albino Wistar rats
Group B: 4000 mg/kg; 10 albino Wistar rats
Group C: 6000 mg/kg; 10 albino Wistar rats
Group D: 8000 mg/kg; 10 albino Wistar rats

Experimental model-
Anti Nephrolithiasis activity model
Animals- Healthy male rats of Wistar strain weighing between 150 and 175 g of equivalent age groups were obtained from central animal house of Institute of Biomedical and Industrial Research, Jaipur. Rats were acclimatized for one month in propylene cages under hygienic conditions and provided with standard animal feed and water ad libitum. All procedures were done in accordance with ethical guidelines for care and use of laboratory animals and were approved by the Institution animal ethical committee (ibir/iaec/02).
Dosage-
Plant extract was suspended in distilled water and was administered (i.p) at doses of 400, 800 mg/kg body weight based on preliminary experimentation.
Experimental procedure-
Ethylene glycol induced hyperoxaluria model was used to assess the antilithiatic activity in albino rats following procedures as under.
Prophylactic regimen (PR) – Animals were divided into five groups containing six animal in each. Group I served as a vehicle treated control and maintained on regular rat food and drinking water ad libitum. All remaining groups (Group II-V) received calculi inducing treatment, comprised of ethylene glycol (EG, 0.4% v/v) with ammonium chloride (NH4Cl, 1% w/v) in drinking water ad libitum for 15 days to accelerate lithiasis. Group III - administered cystone (750 mg/kg body wt.)
Group IV – aqueous extract Capparis decidua (400mg/kg)
Group V- aqueous extract Capparis decidua (800mg/kg),

Group III – V were administered above mentioned doses from day one to day fifteen of calculi induction. Extract and standard drug were suspended in distilled water and given intraperitoneally once daily.

Curative regimen- Animals were divided into five groups containing six animals in each. Group I served as a vehicle treated control and maintained on regular rat food and drinking water ad libitum. All remaining groups (Group II- V) received calculi inducing treatment, comprised of ethylene glycone (EG, 0.4% v/v) with ammonium chloride (NH4Cl, 1% w/v) in drinking water ad libitum for fifteen days to accelerate lithiasis, followed by only EG (0.4% v/v) for next thirteen days.
Group III - administered cystone (750 mg/kg body wt.)
Group IV – aqueous extract Capparis decidua (400mg/kg)
Group V- aqueous extract Capparis decidua (800mg/kg),
Group III – V were administered above mentioned doses from day sixteen to day twenty eight of calculi induction respectively. Extract and standard drug were suspended in distilled water and given intraperitoneally once daily.
After the treatment, the rats were placed in metabolic cages and urine was collected in a glass bottle having 20 µl of 20 % sodium azide as a preservative for twenty four hour. The urine was frozen at - 200C and used for determination of alkaline phosphate (ALP) and lactate dehydrogenase (LDH) and creatinine content. The rats were anaesthetized with diethyl ether and sacrificed by decapitation after twenty four hour of above treatment. Before sacrificing, the blood was taken from orbital sinus into centrifuge without anticoagulant and allowed to clot at room temperature to collect serum. Urine from urinary bladder was directly obtained by puncturing with a needle (5/8 in.) attached to a 1ml tuberculin syringe. After dissection both kidneys were removed and transverse section from both the kidneys was fixed for histological analysis.

Biochemical assays in urine and serum- Serum urea level was estimated by diacetylmonoxime method. The creatinine in both serum and urine was estimated by the method of Bonsnes and Tauskey. Creatine clearance was calculated. Urinary LDH was measured by decrease in absorbance at 340 nm resulting from the oxidation of NADH. The activity of ALP was determined by measuring the conversion of p-nitrophenyl phosphate to p-nitrophenol at 405 nm.

Histopathological studies- transverse sections of kidney tissue were fixed in formaldehyde (10%). The tissue were then dehydrated and embedded in paraffin wax. The paraffin sections (8µ) were then cut and stained in Hematoxylene & Eosine staining and viewed under light microscope. (Anshu Aggarwal et.al)
Observation
Toxicity Study of aqueous extract of Capparis decidua
Group A: No behavioral change and mortality rate observed.
Group B: 10 % mortality rate observed.
Group C: 30% mortality rate observed.
Group D: 50% mortality rate observed.
Result:
Aqueous extract of Capparis decidua (AECD) found no behavioural change and mortality at dose 2000mg while at 4000, 6000 and 8000 mg/kg, 10%, 25%, 50% respectively mortality was observed.
According to Table 1 and Table 2, test drug at both doses shows nephrolithiatic activity against ethylene glycol induced nephrolithiasis in albino wistar rat with comparison to standard drug cystone.
In preventive and curative regimen test sample show significant changes with comparison of hyperoxaluria group in Urinary LDL (Units/ml/mg prt), Urinary ALP (IU/L), Serum Urea (mg/dl) and Serum Creatinine (mg/dl).
In preventive regimen AECD 800 mg/kg (4.76ml/min, 57.35% relief) show greater Creatinine clearance then standard drug cystone (4.07 ml/min, 34.54% relief) and in curative regimen cystone (5.14 ml/min, 125.33% relief) has greater effect then AECD800 mg/kg (3.25 ml/min, 42.48% relief).
Discussion and Conclusion:
A kidney stone is like a small rock that forms in the kidney. Stones form when certain chemicals in the body clump together. A stone can either stay in the kidney or travel through the urinary system. Very small stones might pass through the urinary system without causing much pain. Larger stones can block the flow of urine if they get stuck in the ureters or urethra. Kidney stones do not usually cause any symptoms until they start to pass. The study was undertaken to investigate the anti nephrolithiasis activity of Capparis decidua. In preventive and curative regimen test sample show significant changes with comparison of hyperoxaluria group in Urinary LDL (Units/ml/mg prt), Urinary ALP (IU/L), Serum Urea (mg/dl) and Serum Creatinine (mg/dl).
In preventive regimen AECD 800 mg/kg (4.76 ml/min, 57.35% relief) show greater Creatinine clearence then standard drug cystone (4.07 ml/min, 34.54% relief) and in curative regimen cystone (5.14 ml/min, 125.33% relief) has greater effect then AECD800 mg/kg (3.25 ml/min, 42.48% relief).
Reference –
? Aggarwal A, Singh SK, Gandhi M and Tandon C (2011), Preventive and curative effects of Achyranthes aspera linn. Extract in experimentally induced nephrolithiasis; Indian Journal of Experimental Biology, Vol. 50, March 2012 pp. 201-208
? Nayak BS, Marshall JR and Isitor G (2010), Wound healing potential of ethanolic extract of Kalanchoe Pinnata Lam. Leaf – A preliminary study; Indian Journal of Experimental Biology, Vol. 48, pp 572-576.
? Lieske JC, Haung E and Toback FG (2000), Regulation of renal epithelial cell affinity for calcium oxalate monohydrate crystals, Am. J. Physiol. Renal Physiol 278: F130-F137.
? Rai MK (1994), Herbal medicines in India; retrospect and prospect, Fitoterapia, 65: 483-91.
? Pattewar SV and Pinnata K (2012), Phytochemical and Pharmacological profile, Scholar Science Journals Vol. 2, pp 1-8.
? Sharma A, Chakraborti KK and Handa SS (1991), Antihepatotoxic activity of some Indian herbal formulations as compared to silymarin, Fitoterapia, 62 : 229-35.
? Sharma A, Chakraborti KK and Handa SS (1991), Antihepatotoxic activity of some Indian herbal formulations as compared to silymarin, Fitoterapia, 62 : 229-35.
? Biswas SK, Chowdhury A and Das J (2011), Literature review on pharmacological potentials of Kalanchoe pinnata (Crassulaceae), African Journal of Pharmacy and Pharmacology, vol. 5(10), pp 1258-1262.

CLAIMS:
1. A method of making of pharmaceutical composition of Aqueous extract of Capparis decidua (AECD) comprising the steps of:
collecting Capparis desidua from it origin and drying to powered form;
mixing the powdered form with a sufficient volume of distilled water and extracted with a soxhlet apparatus in a solvent;
Removing the solvent and extract is dried in an oven and weighted.

2.The method as claimed in claim 1, wherein said powdered form with a sufficient volume of distilled water and extracted with a soxhlet apparatus for 16 to 18 hours.
3. The method as claimed in claim 1, wherein said extract is dried in an oven with the temperature of 50°-60°C.
4. A pharmaceutical preparation of Aqueous extract of Capparis decidua (AECD) comprises 800 mg/kg (4.76 ml/min, 57.35% relief) show greater Creatinine clearence then standard drug cystone (4.07 ml/min, 34.54% relief) and in curative regimen cystone (5.14 ml/min, 125.33% relief) has greater effect then AECD800 mg/kg (3.25 ml/min, 42.48% relief).
5. The pharmaceutical preparation of Aqueous extract of Capparis decidua (AECD) as claimed in claim 4, wherein said preparation is used in antinephrolithiasis activity.