IN VITRO INHIBITION OF UROLITHIASIS USING TERPENE … · Rowatinex has been us ed in the treatment of kidney stone after extracorporeal shock. The capsule which consist of an active

IN VITRO INHIBITION OF UROLITHIASIS USING TERPENE ISOLATED FROM SCOPARIA DULCIS.

Abirami Rm#1

, Parimala B#2

, Swathipriya R#3

, Sethuram M*4

, #Department of Biomedical

Engineering, Vel Tech Multi Tech Engineering College, Avadi, Chennai, India. #1

[email protected] #2

[email protected] #3

[email protected] *4

Assistant Professor *Department of Biomedical Engineering,

Vel Tech Multi Tech Engineering College, Avadi, Chennai, India.

*4 [email protected]

Abstract— Urolithiasis is the vital challenge in present

scenario of medical world. About one in 20 people are

affected by kidney stone in their life time. More than

80% of kidney stones are calcium oxalate stones.

Scoparia dulcis, the herbal plants are used in the

treatment of urolithiasis by ancient tribes. Scoparia

dulcis plants consist of major constituents of terpenes

that have the adverse effect on the urolithiasis. Present

study to evaluate the antilithiatic potential of Scoparia

dulcis under in vitro condition. Calcium oxalate stones

were synthesized using in vitro techniques. The

extracted sample and the calcium oxalate were

characterized using FTIR analysis. Terpenes were

isolated using column chromatography and analyzed

through Thin Layer Chromatography. The study of

release kinetics of calcium oxalate through Scanning

Electron Microscope analysis reveals that terpenes

from Scoparia dulcis inhibited the calcium oxalate by

morphological changes with reduction in its size. The

in vitro study of calcium oxalate with terpenes affirms

the percentage of its affinity to dissolve the stones,

which regulates further techniques. There is a great

need for recurrence prevention that requires a better

understanding of the mechanisms involved in stone

formation to facilitate the development of effective

drugs.

Keywords—Terpene, Synthesis, Calcium oxalate,

Column chromatography, Thin Layer

Chromatography, FTIR, SEM.

I. INTRODUCTION

Scoparia dulcis is the widely used traditional

medicinal plant which belongs to the family

scorphulariaceae. It is branched green color, leafy, odor

taste and tough. It is commonly called as sweet broom

weed, liquorice weed and it is locally named as

Sarokkotthini in Tamil, Kallurukki in Malayalam, and

Ghoda Tulsi, Rice Weed in other languages. It is a

perninal herb, which is distributed widely in tropical and

subtropical Indian regions and it grows upto 1m, which

is small, 3 leaves whorled, the stamens and ovary are in

green color, the petioles and pedicles are in 9mm. The

plant has an rich sources Flavones, Terpenes, Steriods

Phenols, Tannis, Saponins, Aminoacids, Coumarins,

Carbohydrates of a biochemical compound. The Fresh

and dried plants has been used for treating diseases. It is

meant famous in treating the kidney stones. The

terpenoids in the herb are responsible for the medicine

effects. Terpenes are common in plants and essential oils. It is

the subunits of isoterpene biosynthetically. The types of

trepenes are evaluvated as Hemiterpenes,

Monoterpenes, Diterpenes, Sesterterpenes,

Sesquiterpenes, Triterpenes, Tetraterpenes,

Polyterpenes, Norisoprenoids. Terpenes are been

International Journal of Pure and Applied MathematicsVolume 119 No. 15 2018, 551-563ISSN: 1314-3395 (on-line version)url: http://www.acadpubl.eu/hub/Special Issue http://www.acadpubl.eu/hub/

551

restricted to a basic aromatic hydrocarbon. They are

volatile and non-polar compound. The importance of

terpenes in the biomedical field use in the insect

repellants, preparation of perfumes & also in cosmetics,

it has a property of antimicrobial, anti-tumour activity,

anti-hyperglycemic activity, anti-inflammatory and in

neuro psychological disorder treatments. Rowatinex has

been used in the treatment of kidney stone after

extracorporeal shock. The capsule which consist of an

active ingredients such as Anethol, Borneol, Camphene,

Fenchone and Pinene. Kidney stone are solid crystalline mineral deposition

occurs in kidney. Size of kidney stone plays major role

in treatment of kidney stone .the passage of stone

through ureter causes severe pain in abdomen, nausea

and haematuria. The causes of kidney stone depend on

various parameters like gout, hyper calciuria, hyper

thyroids, hyper oxaluria, diabetes, hypertension, obesity,

dietary factors, medications and hereditary factor have a

higher risk of kidney stone formation. The treatment

various depending on size of renal calculi deposited, age

of patient and their diseases. The patients may prone to

multiple attacks by kidney stone. There are several types

of kidney stones, calcium oxalate and calcium

phosphate forms 80% kidney stone.

II. MATREIALS AND

METHODS

A. Collection of Samples

The plant specimens were collected from the road side

of Ambattur and Thiruvallur in Tamilnadu during the

month of December. By the side of the road, abundant

population of the plant Scoparia dulcis was present.

Leaves of Scoparia dulcis plants were separated,

washed carefully 2-3 times with running tap water,

rinsed with distilled water, air dried for 1 hour, and

shade dried. The air dried samples were shade dried for

a period of one week. They were coarsely ground in to

powder and preserved in air tight containers. The

sample was then stored room temperature.

B. Solvent Extract Preparation

The extract of the samples were prepared by soaking

100gm of dried powder in 200ml of various solvents for

48 hours. Solvents such as hexane, ethyl acetate and

water were selected. About 200mlof the solvent was

added to the dried sample in a conical flask. The flask

was then covered with cotton wool. After 48 hours the

sample mixture was filtered using a muslin cloth and

then through Whatman No.1 filter paper. The filtrate

was then concentrated by using a rotary evaporator. The

extract was stored and photochemical tests were

analyzed for the presence of terpenes.

C. Qualitative Analysis

Qualitative analysis was carried out by using various phytochemical screening methods

Test For Terpenes Salkowski test: 2 ml of the extract of the leaves, flowers and seeds was mixed with 2ml of extract was mixed

with 2ml of chloroform and 3ml of concentrated

sulphuric acid was added, the appearance of reddish brown color of interface indicates the presence of

terpenes.

Test For Diterpenes Copper Acetate Test: 5 ml of extract were dissolved in

water and treated with 10 drops of copper acetate

solution, formation of emerald green color indicates

presence of diterpenes

International Journal of Pure and Applied Mathematics Special Issue

552

D. Column Chromatography

Column chromatography is one of the most useful

methods for the separation and purification of both

solids and liquids. The principle of column

chromatography is based on differential adsorption of

substance by the adsorbent. This is a solid - liquid

technique in which the stationary phase is a solid and

mobile phase is a liquid. The stationary phase, a solid absorbent Silica gel of

about 230-400 mesh was used to prepare the slurry. In a

separate beaker, solvents mixture of hexane and ethyl

acetate was measured approximately one and a half

times the volume of silica. Silica gel was added to the

solvent, a little at a time by constant swirling. Thus

slurry of silica gel was prepared which was ready to

load in the chromatography column. The packed slurry

was about 10 to 12 cm about the height of the column.

The sample was loaded onto the silica gel column. The sample added was about 2cm about the height of

the column. The sample was allowed to elute on to the

silica bed. When the sample loaded was completely

eluted on to the silica bed, the solvent mixture was

loaded in the column. The eluent was collected in a

series of test tubes. The flow rate of eluent was

maintained at optimum flow rate for each particular

separation. Once the composition of each fraction was

known, the fractions containing the desired compounds

were combined. The TLC plates were examined under

the Ultra Violet cabinet to inference the spots. The spots

were carefully marked for the Retention factor

calculations. Based

on the value of retention factor, similar compounds were collected together.

E. Thin Layer Chromatography

TLC plates used were purchased as 5 cm x 20 cm

sheets. Each large sheet was cut horizontally into plates which are 10 cm tall and 5cm width.

A large beaker with a glass lid was chosen. The

solvent such as the hexane and ethyl acetate were chosen in the ratio of suitable concentration. The solvent were poured into the chamber to a depth of just less than 0.5 cm. The microcap was dipped into the solution and then gently touched the end of it onto the proper

location on the TLC plate. The prepared plates were placed in the developing beaker, the beaker was covered with the watch glass, and it left undisturbed on the bench top. The plate was removed from the beaker and

immediately marked the solvent front with a pencil. The plate was allowed to dry in air. The spots were analyzed under the Ultra Violet cabinet at a wavelength range of about 254 to 366 nm. The mobile phases were traced by spots indicated by pink color. This coloration of spots

indicates the presence of terpenes. Based on the value of retention factor, similar compounds were collected

together. The Rƒ value was calculated according to this

equation:

Rƒ = distance of sample / distance of solvent.

F. Evaluation Of Release Kinetics -

Procedure for synthesis of calcium oxalate

0.5g of Calcium pieces were weighed in a weighing boat and then transferred the pieces to a 250 ml dry

beaker. Very slowly 10 ml of 6M Hydrochloric acid was added to the beaker. The Calcium was dissolved and diluted the sample to 150 ml with distilled Water. This aqueous solution was heated to incipient boiling on a

hot plate in the fume hood. The Ammonium Oxalate solution was added to the hot Calcium Chloride solution


553

and 3 to 4 drops of Methyl Orange was added. 6M of

NH3 solution was added as 1 ml at a time until the

indicator is intermediate between Orange and yellow or

until a persistent precipitate appears. A gravity funnel was set-up in a Ring and filtered using a fine filter paper to filter the bulk of the liquid from the solution. A few squirts of Water used to wash the solid and the beaker.

A rubber policeman was used to completely transfer the remaining solid to the funnel. The solid was washed with five portions of 7 ml cold Water. The filter paper is removed from the funnel and spread it out on a watch glass so that the solid can dry.

Observation-Formation of white crystalline compound that infers the calcium oxalate stone.

G. Examination of release kinetics of calcium

oxalate in Terpenes - In Vitro Anti Lithiatic Activity

Test

The dissolution percentage of calcium oxalate was

evaluated by taking exactly 1 gram of calcium oxalate at

different time intervals in the organic extraction

terpenes. The crystallization inhibition effect of terpene

extracted

from Scoparia dulcis was estimated by course of time

measurement of turbidity changes, due to the

crystallization effects of calcium oxalate. The organic

extract terpene isolated from the plant was chosen at

different time intervals in order to examine the release

kinetics of calcium oxalate in the terpenes. Three vital

extraction variables are examined such as time duration,

temperature and drug-solvent release kinetics. The

terpene turbidity test was carried out under the constant

temperature of about 37°C.

H. FTIR Analysis Fourier transform infrared spectroscopy is used to

analyse the chemical constituents by obtaining the

infrared spectra from various interference patterns. The

organic extract and calcium oxalate was characterized

by using Fourier transform infrared spectroscopy to

study the preliminary chemical constituents based on the

transmission versus wave number infrared spectra

I. SEM Analysis

Scanning Electron Microscopy is used to study the

morphological characteristics of various materials. The

in vitro synthesis of calcium oxalate and the calcium

oxalate suspended in the terpene from scoparia dulcis

were analyzed through SEM analysis. The

morphological changes of the calcium oxalate with

various time durations in presence of terpene were used

as the preliminary analysis test to study the nature and

morphology of calcium oxalate.

III. RESULTS AND DISCUSIION

The calcium oxalate crystals that have been produced

in this in vitro study were similar to the crystals in the

urine of patient with calcium oxalate crystals. In this

study hexane extract of scoparia dulcis has been used

since many of the active compounds of terpenes in the

plants, were dissolved in it and extracted through

solvent extraction method. Because the polarities of

three different solvents like hexane, ethyl acetate and

aqueous are different, the active compound in them is

also different. Terpenes were isolated using the column

chromatography with various concentrations of solvents

such as hexane and ethyl acetate. The solvents in the

ratio 4:1 were proven to be the most effective in terms

of terpenes isolation. The isolated components were

analyzed through Thin Layer Chromatography

techniques under the Ultra Violet cabinet to infer the

presence of terpenes. The analysis of various fractions

provided the different Rf values which was shown in

table 1. The results of the in vitro inhibition effects of

different concentrations of terpenes from Scoparia

dulcis, on the area of calcium oxalate crystal formation

were shown in the table 2.


554

FTIR analysis on organic extract FTIR analysis of extract of Scoparia dulcis shows peaks

at 2924 cm-1

, 2950 cm-1

, 2405 cm-1

, 1734 cm-1

, 1589

cm-1

, 1361 cm-1

, 1222 cm-1

, 1011 cm-1

, 665 cm-1

.FTIR

analysis of organic extract shoes peaks that represent the

presence of These peaks represent the functional groups

such as nitro groups, acids, alkyl halides, alcohols,

cyano group, amines and hydrocarbons.

FTIR Analysis on calcium oxalate FTIR analysis of calcium oxalate indicates the

presence of its chemical constituents with presence of various bonds. The FTIR analysis graph have two peaks

at 775 cm-1

and at 658 cm-1

as a off- water bending band. Two transmission bands were observed at 1611

cm-1

and 1314 cm -1

. The first is attributed to the stretching band anti symmetric carbonyl and second is symmetric stretch band, this two bands emphasis on the formation of calcium oxalate.

SEM Analysis SEM (Scanning electron microscopy) analyses the

surfaces of materials, particles and fibers so that fine

details can be measured and analyzed via image

analysis. Also SEM analysis attest the morphological

changes in the calcium oxalate suspended in terpene.

The morphology of the calcium oxalate is crystalline in

nature. Micro scale study at the size of 10 micrometer

and magnification at 6.0K was used as the basic study

tool. Thus crystalline nature of the calcium oxalate get

truncated and eroded when it dissolves in the terpene

from scoparia dulcis. This provides difference between

the two Calcium oxalate stones. Fig.1 displays a

crystalline structure of calcium oxalate and Fig.2

displays the amorphous structure of calcium oxalate. As

the duration increases, weight of the stone decrea


555

TABLE I Retention factor values of fractions under TLC

Screening.

MOBILE

FRACTIONS PHASE Rf VALUE

[Solvents]

1 0.20

2 0.30

3 0.44

4 Hexane : Ethyl 0.50

5 acetate 0.67

[4:1]

6 0.80

7 0.76

8 0.81

9 0.73


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In vitro Analysis of calcium

oxalate TABLE II

Effects of terpenes of Scoparia dulcis on calcium

oxalate with various time durations.

WEIGHT WEIGHT

OF OF

SAMP DURATIONS CALCIUM CALCIUM

LE (hours) OXALATE OXALATE

(Initially in (In

gram) Terpene)

1 24 1 0.90

2 48 1 0.87

3 72 1 0.83

4 96 1 0.75

5 120 1 0.69

Fig.2 FTIR Analysis of in vitro synthesized calcium oxalate.


557

FTIR Analysis

SEM Analysis

Fig.1 Analysis of Calcium oxalate. Fig.1 FTIR Analysis of leaves sample of Scoparia dulcis


558

Fig.2 Analysis of Calcium oxalate suspended in terpene.

III. CONCLUSION

Evaluation of release kinetics of calcium oxalate

infers the effective dissolving concentration of terpenes.

The test to study the dissolving ability of the kidney

stone reveals the percentage of its affinity to dissolve

the stones, which regulates further techniques. The

product yield from the organic extracted from Scoparia

dulcis can be used as the oral solution to dissolve the

calcium oxalate. Further study the effect of compounds

in Scoparia dulcis extract on urine of urolithiatic

patients to see how effective it on crystals of lithiatic

patients and also clinical trials are needed on the human

beings with calcium oxalate urolithiasis. There may be a

potential for using alternative medicine for patients with

urolithiasis apart from surgery and western medicines.

ACKNOWLEDGEMENT

We, the students of VEL TECH MULTI TECH

ENGINEERING COLLEGE from the department of

BIOMEDICAL ENGINEERING, are extremely grateful

to company for the confidence bestowed in us and

entrusting our project paper, we also extend our

gratitude to project guide Mr.M.Sethuram, faculty who

assisted us in compiling the project, we would also like

to thank all the faculty members of our department for

their advice, motivation & guidance.

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IN VITRO INHIBITION OF UROLITHIASIS USING TERPENE … · Rowatinex has been us ed in the treatment of kidney stone after extracorporeal shock. The capsule which consist of an active