INTRODUCTION:

However, in addition to the traumatic effects of shock waves, persistent residual stone fragments and the possibility of infection, suggest that ESWL may cause acute renal injury, a decrease in renal function and an increase in stone recurrence(Begun, F.P. 1991). Urolithiasis is still a mysterious disease even after extensive research in urology. Sophisticated instruments, investigation etc., have failed to trace out the exact mechanism of urolithiasis, but they are manifesting this condition. The treatment in modern medicine is not only expensive but also not easily affordable to the needy poor.

Calcium stones:

Hypercalciuria

Hypocitrauria

Uric acid stone

METHODOLOGY:

Preliminary phytochemical analysis:

Test for alkaloids:

Mayer’sTest (Potassium Mercuric Iodide)

Dragondroff’s Test (Potassium Bismuth Iodide)

Wagner’s Test (Iodine in Potassium Iodide)

Hager’s Test (Picric acid Test)

Test for carbohydrates:

Molisch’s Test

Fehling’s Test

Barfoed’s Test

Test for proteins:

Millon’s Test

Ninhydrin Test

Biuret Test

Xanthoprotein Test

Test for tannins and phenolic compounds:

Ferric Chloride Test

Gelatin Solution Test

Test for phytosterols and terpinoids:

Salkovaski Test

Libermann – Burchard Test

Test for glycosides:

Borntragers Test

Modified Borntragers Test

Keller – Killiani Test

Legal’s Test:

Baljet’s Test

Test for Flavanoids:

Test for Saponins:

Test for Steriods:

Test for Terpenoids:

ANTIUROLITHIATIC ACTIVITY STUDY:

Acute toxicity studies:

The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Cooperation and Development (OECD) received from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). One-tenth of the median lethal dose (LD50) was taken as an effective dose.

Ethylene glycol induced urolithiasis model:

Ethylene glycol induced hyperoxaluria model was used to assess the antilithiatic activity in albino rats. Animals were divided into nine groups containing six animals in each.

Animal selection:

Healthy Inbred Albino rats of Wistar strain, of male, aged around 2 to 3 months and weighing 150-200 g were selected for the antiurolithiatic activity used. The animals were acclimatized to standard laboratory conditions (temperature: 25±2°C) and maintained on 12-h light-dark cycle, relative humidity of 45-55%, and maintained on 12–hour light: 12–hour dark cycle in animal house. They were provided with regular rat chow (Lipton India Ltd., Mumbai, India) and drinking water ad libitum. The animal care and investigational protocols were in accordance with Institutional Animal Ethical Committee (IAEC).

Treatment protocol:

The grouped animals received the treatment as follows

Group 1. Received normal diet and served as controls.

Group 2. Lithiatic control: The animals were given normal diet and 1% Ethylene glycol in drinking water, for 28 days.

Group 3. Received 1% ethylene glycol in drinking water and then treated with Ethanolic extract of GV at a dose of 200mg/kg orally, for 28 days.

Group 4. Received 1% Ethylene glycol in drinking water and then treated with Aqueous extracts of GV at a dose of 200mg/kg orally, for 28 days.

Collection and analysis of urine:

All animals were kept in individual metabolic cages and 24 h urine samples were collected on 14^th, and 28^th day of calculi induction treatment. The volume and calcium content of urine were measured. Calcium in urine was estimated using kit by COBAS MIRA PLUS auto analyzer. Urine was analyzed for oxalate, magnesium, phosphate, uric acid, citrate and total protein (Martino Mangraella 2000, Ross Morton A 2002, Prasad K.V, 1993)

Serum analysis:

The blood was collected from the retro-orbital sinus under anaesthetic condition and serum was separated by centrifugation at 10,000rpm for 10 min and analyzed for creatinine and uric acid. The creatinine kit (Reckon Diagnostics Pvt. Ltd., India) and uric acid diagnostic kit (Span Diagnostics Ltd., India) were used to estimate serum creatinine and uric acid levels respectively.

ANTIOXIDANT ACTIVITY:

Renal cellular exposure to oxalate (Ox) and/or CaOx crystals leads to the production of Reactive Oxygen Species (ROS), development of oxidative stress followed by injury and inflammation. Renal injury and inflammation appear to play a noteworthy role in stone formation. An overproduction of ROS and a reduction in cellular antioxidant capacities, due to down-regulated expression of the antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glucose-6 phosphate dehydrogenase) as well as radical scavengers (vitamin E, ascorbic acid, abridged glutathione) leads to the development of Oxidative Stress (OS) (Ying, W. M, 2002) .Oxidative stress followed by renal cell injury and inflammation due to lipid peroxidation (Udupa. K.N., Singh R.H.1995). Loss of membrane integrity subsequently facilitates the retention of calcium oxalate crystals and growth of stones in renal tubules. Recent studies have provided evidence that CaOx kidney stone patients malondialdehyde (MDA) in their urine, indicating ROS in kidneys of CaOx stone patients. Urinal excretion of these MDA is considered as a marker of renal epithelial cell injury.

DPPH radical scavenging activity:

The free radical scavenging activity was measured by (Kumaran A 2007) method, the decrease in absorbance of Ethanolic solution of DPPH. A stock solution of DPPH (33mgL^-1) was prepared in methanol and 5ml of this stock solution was added to 1ml of the plant extract solutions at different concentrations (25, 50, 75, 100, 150, 200, 250, 2500ug/ml^-1). After 30min, absorbance was measured at 517nm and compared with the standard ascorbic acid (10-50ugml^-1) pH 7.4. Percentage of DPPH scavenging activity of the plant extracts and the standard was calculated. The percentage extract of inhibition was calculated by the formula [(Ao-A1)/Ao] x100, when Ao is the absorbance of the control and A1 is the absorbance of the extract/standard.

Assay of nitric oxide scavenging activity:

% NO radical scavenging activity =

(control OD - sample OD / control OD) × 100.

RESULT AND DISCUSSION:

Table 2. Estimation of Extractive values and Ash values

S. No.	Parameters	GV
S. No.	Parameters	(% w/w)
1	Extractive Values
a.	Petroleum ether	16.23
b.	n-hexane	3.3
c.	Chloroform	18.63
d.	Methanol	20
e.	Water	13.63
2	Ash Values
a.	Total Ash	6.35
b.	Acid insoluble Ash	2.54
c.	Water soluble Ash	1.43
d.	Sulphated Ash	2.13
3	Loss on Drying	0.89
4	Crude fibre content	10.2

The Phytochemical screening results are as follows

Table 3. Phytochemical screening

S. No	Chemical Test	GV Aqeous Alcohol
(1)	Alkaloids
A	Mayer’s test	+	+
B	Dragendroff’s test	+	+
C	Wagner’s test	+	+
D	Hager’s test	+	+
(2)	Carbohydrates
A	Molisch’s test	+	+
B	Fehling’s test	+	+
C	Barfoed’s test	+	+
(3)	Proteins and Free Amino acids
A	Ninhydrin test	_	-
B	Biuret test	+	+
C	Xantho Protein test	+	+
(4)	Tannins and Phenolic Compounds
A	Ferric chloride test	+	+
B	Lead acetate test	+	+
C	Gelatin test	+	+
(5)	Phytosterols
A	Libermann Burchard test	-	+
B	Salkowski test	-	+
(6)	Flavanoids
A	Shinoda test	+	+
(7)	Saponins	+	+
(8)	Glycosides	-	-
9	Terpenoids

Antiurolithiatic Activity:

In the present study, the urine amount augmented in the treated group’s animals than that of the control and it abridged in the untreated lithiatic animals when comparing to the standard and urinal concentration of the an assortment of ions investigated varied drastically, subsequent ethylene glycol treatment in the lithiatic control. The oxalate, calcium, uric acid, creatinine and phosphate excretion were notably increased on day 14^th& 28^th respectively for GROUP-II following ethylene glycol treatment. Management with Ethanolic and aqueous extracts of Glochidion velutinum abridged the excretions notably on 14^th day of treatment and supplementary abridged on 28^th day.

Table 4. Effectof Glochidion velutinum on urinal output in urolithiasis induced rats.

Days

Group-I

Group -II

Group-III

Group-IV

7.25±

0.52

7.30±

0.60

7.35±

0.66

7.90±

0.70

7.89±

0.60

5.35±

1.36

8.45±

1.50

8.20±

1.20

7.56±

0.76

4.95±

1.60

8.65±

1.86

8.75±

1.30

Table 5 Effect of Glochidion velutinum on serum Biochemical parameters on 28day

Groups

Mag-nesium

(mg/dl)

Calcium

(mg/dl)

Uric

acid

(mg/dl)

Creati-nine

(mg/dl)

Oxalate

(mg/dl)

Phos-phate

(mg/dl)

Group-

4.80±

0.86

9.40±

1.32

3.45±

0.40

0.56±

0.03

6.6±

0.57

12.06±

1.43

Group–

1.38±

0.25

18.30±

2.34

9.7±

1.10

1.01±

0.13

12.60±

1.61

26.01±

3.25

Group–

III

3.28±

0.46

11.85±

1.88

4.55±

0.55

0.91±

0.09

8.45±

0.88

20.10±

2.65

Group–

3.67±

0.52

11.22±

1.60

4.10±

0.46

0.80±

0.07

8.12±

0.78

19.85±

2.05

GP₁- Normal; GROUP-II- Lithiatic Control, GP₃- EEGV (200mg/kg), GP₄- AEGV (200mg/kg),

GP- Cystone herbal tablets (100mg/kg)

Histopathological study:

Fig 8. Section of kidney glomerului-I

Fig 9. Section of kidney glomerului-II Fig 10. Section of kidney glomerului-III Fig 11. Section of kidney glomerului-IV

In vivo antioxidant activity:

Table 8. Effect of Aqueous and Ethanolic extracts of Glochidion velutinum on antioxidant enzymes in renal tissue

Treatment	Catalase A/protein	SOD B/mg protein	GSH nmoles/mg protein	MDA nmoles/mg protein
Normal control	3.20±0.18	9.20±0.18	3.72±0.16	3.88±0.28
Ethylene glycol	0.98±0.02	3.68±0.07	0.52±0.06	10.05±0.46
Cystone	2.80±0.16	7.05±0.11	2.68±0.14	4.20±0.30
AEGV 200mg/kg	2.32±0.09	6.55±0.10	2.12±0.10	5.68±0.35
EEGV 200mg/kg	2.28±0.08	6.60±0.12	2.20±0.11	5.76±0.45

· Values are expressed as Mean± SEM

· Values were found out by using ONE WAY ANOVA Followed by Newman keul’s multiple range tests.

· **(a)values were notably different from normal control (GP₁) at P< 0.01

· **(b) values were notably different from Lithiatic control (GROUP-II) at P<0.01