The document summarizes an in-vitro study evaluating the antilithiatic (anti-kidney stone forming) and antioxidant activity of leaves of Elytraria acaulis. It includes details on the objective to evaluate the leaves' ability to inhibit calcium oxalate crystallization and growth using various assays. The methodology describes preparation of an aqueous extract of the leaves and testing the extract in assays for calcium oxalate nucleation and aggregation, calcium oxalate and brushite crystal growth, and various in-vitro antioxidant activity assays including DPPH radical scavenging and reducing power. The results section presents some standardized parameters of the crude drug and extract including morphological characteristics, physicochemical properties, and extractive

1. In-vitro Evaluation of Antilithiatic and
Antioxidant Activity
Submitted By
Sonika Chouhan
M.Sc. (Biotechnology)

2. Contents:
Introduction
Literature review
Disease profile
Objective plan of work
Material and method
Results
Urolithiasis (Kidney stone)

3. Introduction
• India is known to be rich repository of medicinal plants. Ayurveda is
widely practiced in India.
• In recent years, the use of plants in traditional medicine has increased
the interest in ethno-botanical studies throughout the world.
• In fact, World Health Organization (WHO) estimates that 70% of
populations from many countries are using traditional or folk medicine
to cure various ailments [Jiofack et al., 2010].
• Traditional herbal medicines are an important part of the healthcare
system in India [Singh et al., 2010].
• Urolithiasis (nephrolithiasis) or kidney stone is formation of urinary
calculi at any level of urinary tract.
• This study demonstrated that aqueous extract of Elytraria acaulis of
possess a high antioxidant activity and an ability to inhibit the CaOx
crystallization in vitro by using different models,
• Calcium oxalate nucleation and aggregation, Calcium oxalate growth
assay, brushite crystal growth assay in single diffusion gel method.

4. Urolithiasis
• Urolithiasis is known as kidney stone or nephrolithiasis.
• It is the third most common disease.
• A kidney stone is a hard, crystalline mineral formed with in the kidney or urinary
tract.
• It is the formation of urinary calculi at any level of urinary tract.
• It also refers to the formation and solid concentration or crystal aggregation
formed in kidney and retention of solid non metallic dietary minerals (stones) in
the urinary tract.
• It is form when there is decrease in urine volume or an excess of stone forming
substances in urine.
• Dehydration is the major risk of kidney stone.
• It is estimated that 12% of world population experiences renal stone disease with
a recurrence rate of 70-80% in male and 47-60% in female [Mohan, 2010;
Sundararajan et al., 2006].
• Urinary calculi are the third most common affliction of the urinary tract which is
exceeded by the urinary tract infections and prostate diseases [Hamid et al.,
2007].

5. Conti…
• CaOx stones are responsible for the formation of stones in the kidney
[Verkoelen et al., 1995].
• The crystallisation of the CaOx begins with increased urinary
supersaturation, with the subsequent formation of the solid crystalline
particles within the urinary tract.
• This is followed by nucleation, by which stone-forming salts in
supersaturated urinary solution coalesce into clusters, and increase in size by
the addition of new constituents [Basavaraj et al., 2007].
• These crystals grow and aggregate with other crystals in solution, and are
ultimately retained and accumulated in the kidney [Kok et al., 1990].
• Therefore, if this progression of crystallization can be prevented, lithiasis
could also be prevented.
• Recently, Oxidative stress has been identified as another contributing factor
for stone initiation and progression [Huang et al., 2003].

7. Supersaturation Crystallisation Crystalluria
Crystal retention and
stone formation
Promoter
Inhibitor
Figure 1: Pathogenesis of urine stone pathogenesis
Promoters
Calcium
Sodium
Oxalate
Urate
Cystein
Low urine pH
Tamm-Horsfall protein
Low urine flow
Inhibitors
Inorganic
Citrate
Magnesium
Pyrophosphate
Organic
Tamm-Horsfall protein
Urinary Prothrombin fragment 1
Protease inhibitor: inter α inhibitor
Glycosaminoglycans
Osteopontin (Uropontin)
Renal lithostathine
High urine flow

8. Figure 2: Risk of kidney stone

10. Management of Urolithiasis
Non-steroidal anti-inflammatory drugs (NSAIDs)
Opioids
Medical expulsive therapy (MET)

11. Technique for treatment
Open Surgery
Laparoscopic surgery
Ureteroscopy

12. Antioxidant
• An antioxidant is a molecule capable of inhibiting the oxidation of
other molecules.
• Oxidation is a chemical reaction that transfer electron or hydrogen
from a substance to an oxidising agent.
• Oxidation reaction can produce free radicals. In turn these radical
can start chain reactions.
Figure 4: Antioxidant

13. Benefits of antioxidants
• Destroy the free radicals that damage cells.
• Promote the growth of healthy cells.
• Promote cells against premature, abnormal ageing.
• Provide excellent support for the body’s immune system, making it
an effective disease prevention.

14. Oxidative
Stress
Brain
Alzheimer,
Parkinson, Stroke
Skin & Hair
Melanin
abnormalities, grey
hair
Kidney
Urolithiasis, Diabetic
nephropathy
Eyes
Mascular & Retinal
degeneration,
Cataracts
Blood
Thalessemia,
Hypertension
Joints
Rheumatoid arthritis,
Osteoarthritis
Heart
Hypertension ,
Heart failure
Liver & Pancreas
Diabetes, Islet of
Pancreas
Lungs
Asthma, Allergies
Figure 5: Deleterious effect of Oxidative stress on human health

15. Objective & plan of work
Objective
To evaluate antilithatic and antioxidant activity of leaves.
Plan of Work
The study was carried out in following manner.
1.Collection and identification of the leaves.
2.Processing of the leaves.
3.Pharmacognostic and Phytochemical studies
 Microscopy of leaf
 Standardization of the leaves.
 Preparation of extracts of the powered leaves.
 Qualitative chemical analysis of the crude extracts.
 Quantitative estimation of phytoconstituents.
4. In-vitro assessment of antilithatic effect
 Nucleation and aggregation assay (CaOx crystallization)
 Growth assay (Brushite crystals growth in single diffusion gel method)
5. In-vitro assessment of antioxidant activity
 DPPH scavenging activity
 Hydroxyl radical scavenging
 Reducing potential
6. Statistical analysis

16. Preparation of Aqueous Extract of the Leaves
Material & Methods

17. Ten gram leaves (coarse powder)
Weight and kept in thimble
Extracted with 250 mL of
petroleum ether
Soxhlet apparatus (50°C)
Defatting of plant material
Figure 7: Soxhlet
apparatus

18. Extraction by cold maceration
Defatted marc was collected from the thimble
Soaked (100 ml purified water)
48 h (temperature range of 20–26 °C)
1% chloroform added (avoid microbial
growth)
Filtered (Whatman Filter Paper No.1)
After 48 h
Filtered extract dried (rotary
evaporator)
Figure 8: Extraction
by cold maceration

20. 1. Determination of Ash value
(To detect purity of drug)
Total ash value
(To remove all carbon)
Acid insoluble ash
(Gives idea about
earthy matter)
Water soluble ash
(Indicates the pre. of
salts, amt. in-org.
matter)
2. Loss on drying
(loss in wt. due to the
pre. of moisture or volatile
matter)
3. Detemination of
Extractive value
(Pre. of active
constituents)
Alcohal (methanol)
soluble
Water
(Distilled) soluble
4. Determination of
pH value
Figure 9: Ash value Figure 10: Extractive value

21. Total Phenolic
[Singleton and Rossi, 1999]
1mL extract
1 mL folin ciocalteu
reagent (Dil.1:20)
4 mL NaCo3 (75g/L)
10 mL distilled water
Mixture kept in dark
at room temp. (2h)
Centrifuge 2000 rpm
(5 min)
Absorbance (760nm)
Total Flavanoid
[Marinova et al., 2005]
1mL extract
Standard sol.
Quercetine (4mL in
10mL vol.flask)
0.3mL, 5% NaNo2
0.3mL, 10% AlCl3
2mL, 1M NaoH
Absobance (510nm)

22. Total Saponin
[Obadoni and Ochuko, 2002]
Ten gram powder(crude)
50mL 20% Aq. ethanol
Heat (water-bath) 4h at
55°C
Filter and re-extracted
100mL 20% ethanol
Combined extract reduced
to 20mL (water-bath) 90°C
Transfer in 250mL
Seperating funnel
Add 10mL ether layer &
shake
Ether(upper layer) discarded
Purification process repeat
(3 times)
Add 30mL n-butanol
Combined extract washed with
5mL, 5% Aq. Nacl (lower layer)
Remain heat (water-bath)
70°C
Sample dried in Oven at
50°C

23. Assessment of In vitro Antilithiatic
Activity

24. (A) Calcium oxalate nucleation and aggregation :
I. In vitro Crystallization of Calcium Oxalate [Hess et al., 2000]
Nucleation : Increase in
absorbance
Aggregation : Decrease in
absorbance
25mL Naox
Hot plate (magnetic stirrer)
37°C, 800 rpm
1mL extract
25mL Cacl2
Absorbance (620nm)
Reading 5 min (every 15 sec)
Reading 10 min
(every 1 min)

25. (B) Calcium oxalate growth assay
[Chaudhary et al., 2010; Aggarwal et al., 2010]
30mL buffer (Nacl+Tris HCl)
20mL NaoH
20mL Cacl2
1mL extract
600µl COM
Absorbance
(214nm)
1 min

26. II. Brushite Crystals Growth Assay in Single
Diffusion Gel Method
[Joshi et al., 2005(b)]
5mL sodium meta silicate
2.7mL Ortho phosphoric acid
Gelation
10mL Cacl2
Crystal growth appeared in gel
Increase in length of crystals
Treatment (5th day)
Extract (Test sample)
8th day
Observed (Microscope at 10X)

27. Assessment of In Vitro Antioxidant
Activity

28. I. DPPH (1,1-Diphenyl-2-picryl-hydrazil) free radical scavenging
activity [Brand-Williams et al.,1995]
Solution : DPPH in ethanol3.5mL solution
0.5mL extract (different conc.)
Test sample : In water
Mix & shake
Kept at room temp. (30 min)
Absorbance (517nm)
Standard : Butylated
hydroxyl- toulene (BHT)
Lower absorbance indicated higher
free radical scavenging activity

29. II. Scavenging of hydrogen peroxide
[Ruch et al., 1989]
Phosphate buffer saline (pH 7.4)
Hydrogen peroxide solution
(1.5 ml, 40 mM)
Absorbance (230nm)
In 10 min
A solution of hydrogen peroxide (40 mM)
AqEA Extract prepared in distilled water
(1.5ml) in different concentrations
Standard : Ascorbic acid

30. III. Reducing power assay
[Oyaizu, 1986]
1.5mL phosphate buffer
0.5mL extract (different conc.)
1.5mL potassium ferricynide
Incubated 20 min (55 °C)
1.5mL Trichloro acetic acid
Centrifuge 3000rpm (10 min)
1.5mL layer diluted with water
Add Fecl3 (300µl)
Absorbance (700nm)
Increased absorbance indicated
increase reducing power

31. Results

32. Standardization of Crude Drug and Extract
Table 1: Morphology
Morphological
characters
Observation
Color Dark Green
Odour Odourless
Taste Bitter
Size Varying in size
Microscopic Evaluation

33. Table 2: Physiochemical evaluation
Sr. No. Standardization parameters Value %w/w
01 Ash analysis
 Ash content (Total ash)
 Acid in-soluble ash
 Water soluble ash
15.48±0.033
4.417±0.136
6.425±0.216
02 Extractive value (Maceration process)
 Alcohol soluble 17.79±0.844
 Water soluble 20.62±0.791
03 Moisture content (Loss on drying) 4.267±0.145
04 pH
 1% aqueous solution 6.685±0.037
[Values are expressed as mean±SEM; n=3]
Percentage (%) Yield
The aqueous extract of dark brown color and dry amorphous consistency was
obtained with percent yield of 30.21 % w/w.

34. Phytoconstituents Phytochemical Test Inference
Carbohydrates Molish’s test Absent
Proteins Biuret test Present
Xanthoprotein test
Amino acids Ninhydrine test Present
Alkaloids Hager’s Test
PresentWagner’s Test
Mayor’s Test
Glycosides Keller-killiani test Absent
Saponins Foam Test Present
Sterols (Phytosterols) Salkowski test Absent
Tannins and Phenolic
compounds
Ferric chloride test
AbsentLead acetate test
Gelatin solution
Acetic acid test
Flavonoids Shinoda Test
PresentLead acetate test
Table 3: Phytochemical screening

35. Phytoconstituents Value
Total Flavonoids Content
(Quercetin equivalents (mg)/g
of extract)
39.68±3.361
Total Phenolics Content
(Tannic acid equivalents (mg)/g
of extract)
16.60±1.977
Total Saponins (g/100 g
powdered drug)
3.2
Table 4: Phytoconstituents (Quantitative estimation)
[Values are expressed as mean±SEM; n=3]

36. Reference
Previous Research Papers (Urolithiasis)
Indian Medicinal Book