IN VITRO INHIBITION OF UROLITHIASIS USING TERPENE … · Rowatinex has been us ed in the treatment...
Transcript of IN VITRO INHIBITION OF UROLITHIASIS USING TERPENE … · Rowatinex has been us ed in the treatment...
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
Assistant Professor *Department of Biomedical Engineering,
Vel Tech Multi Tech Engineering College, Avadi, Chennai, India.
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
International Journal of Pure and Applied Mathematics Special Issue
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.
International Journal of Pure and Applied Mathematics Special Issue
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
International Journal of Pure and Applied Mathematics Special Issue
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
International Journal of Pure and Applied Mathematics Special Issue
556
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.
International Journal of Pure and Applied Mathematics Special Issue
557
FTIR Analysis
SEM Analysis
Fig.1 Analysis of Calcium oxalate. Fig.1 FTIR Analysis of leaves sample of Scoparia dulcis
International Journal of Pure and Applied Mathematics Special Issue
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.
IV. REFERENCES [1] Meera paul, kavitha vasudevan, krishnaja.k.r,
―scoparia dulcis: a review on its phytochemical and pharmacological profile‖, vol 4, issue 4, 2017.
[2] Jones ofari-amoah, George koffuor, George owsul,
vener n orish, ―Effects of a hydro-ethanolic extract
of scopariadulcis on systemic inflammation and
cachexia inan allergic asthma‖, 2016.
[3] Anju mathew, a. Malar retna, ―Antilithiatic activity
and pharmacognostic studies of scoparia dulcis‖, vol 2, no 1, January 2016.
[4] V.E.Idachristia, R.Senthamarai, ―Qualitative and
quantitative pharmacognostical studies on scoparia
dulcis linn leaf‖, April 2015, vol 3, issue 1. [5] Wankupar wankhar, sakthivel srinivasan, ravindran
rajan, sheeladevi rathinasamy, ―Evaluating in vitro
antioxidant activity and gc-ms analysis of scoparia
dulcis linn‖, (scrophulariacae vol. 5 (07), pp. 029-
034, July, 2015. [6] R. Kachkoula , T. Sqalli Houssainib, Y. Miyahc,
M.Mohimd, R. El Habbanie, A. Lahrichia, ―The
study of the inhibitory effect of calcium oxalate
monohydrate’s crystallization by two medicinal &
aromatic plants‖, 2017 May.
International Journal of Pure and Applied Mathematics Special Issue
559
[7] Parveen Kumar Goyal, Santosh Kumar Verma, Anil
Kumar Sharma, ―Optimization of extraction
protocol of Parmeliaperlata and its validation for protective effects against oxalate‖, induced renal
injury in NRK-52E cells, 2016 September. [8] G. Uma, a. Najila banu1, j. Sathica taj, u. Josephine
benedit bai, ―Phytochemical screening and
antibacterial activity of scoparia dulcis extracts‖, vol 7, issue 3, 2014.
[9] Rupjyoti saikia, anupam das talukdar, pankaj
chetia1and manabendra dutta choudhury, ―Preliminary
phytochemical investigation and TLC profiling of
ethno medicinally important plant, scoparia dulcis
linnaeus pleione‖, Vol 7(2): 366 – 372, 2013. [10] M.r. mishra, r.k. behera, s. Jha, a.k. panda, a.
Mishra1, d.k. pradhan1p.r. choudary, ― A brief
review on phytoconstituents and
ethnopharmacology of scoparia dulcis linn.
(scrophulariaceae)‖, 2011 422-438. [11] Swapna g, jyothirmai t, lavanya v, swapnakumari s,
sri-lakshmi prasanna, ―A extraction and
characterization of bioactive compounds from plant extracts a review‖, Eurj pharm sci res Vol 2(1): 1-6, 2015.
[12] Sheel r., nisha k. And kumar j, ―Preliminary phytochemical screening of methanolic extract of
clerodendron infortunatum‖, 2014. [13] Mir ma, sawhney ss, jassal mms, ―Qualitative and
quantitative analysis of phytochemicals of taraxacum officinale‖, Journal of pharmacy andpharmocology, 2013; 2(1):001-005.
[14] Prohp, T. P., and Onoagbe, I. O, ―Determination of
phytochemical composition of the stem bark of
triplochiton scleroxylon k. schum. (sterculiaceae)‖, International Journal of Applied Biology and
Pharmaceutical Technology, 2012, Volume 3, Issue 2. Pp. 68-76.
[15] Atodariya U, Barad R, Upadhyay S, Upadhyay U, ―Anti-Urolithiatic Activity of Dolichos Biflorus Seeds‖, J Pharm Phytochem, 2013; 2: 209-213.
[16] Abdur R, Afaq SH, ―Study on extracts on Permalia
perlata Ach. For its antimicrobial potential against certain micro-organisms‖, Int Res J Pharm, 2013, (4)11:102-6.
[17] Sumayya sikandari, Pratima Mathad, ―In vitro anti urolithiatic activity of Butea monosperma lam & Nigella sativa linn‖, Annals of Phytomedicine, 2015; 4(1): 105-107.
[18] Narendra Vyas, Ameeta Argal, ―Anti urolithiatic
activity of extract and Oleanolic acid isolated from
the roots of Lantana camara on Zinc disc
implantation induced urolithiasis‖, ISRN
Pharmacology, 2013; 1-5 [19] Gracelin, D.H., Britto, A.J and Kumar,
B.J.R,
―Qualitativeandquantitativeanalysisof Phytochemicals in five Pteris species‖, International Journal of Pharmacy and
Pharmaceutical Sciences, 2012, Vol 5 Sppl. Pp: 105-107
[20] Ashok Kumar Bagepalli Srinivasa, Kuruba L, Khan S and Saran GS, ―Antiurolithiati Activity of
Gokhsuradi Churan, an Ayurvedic formulation by
In Vitro Method‖, Advanced Pharmaceutical Bulletin, 2013; 3(2):477-
479.
[21] Moldenke HN, ―Notes on the genus
Clerodendrum
(Verbenaceae)‖, Phytologia. 1985; 57: 334-365 [22] Steane DA, Scotland RW, Mabberley DJ, Olmstead
RG, ―Molecular systematics of Clerodendrum (Lamiaceae): its sequences and total evidence‖, American Journal of Botany, 1999; 86: 98-107.4.
[23] D.G.Barceloux, ―Medical Toxicology of
Natural
International Journal of Pure and Applied Mathematics Special Issue
560
Substances: foods, fungi, medicinal herbs, plants,
and venomous animals‖, John Wiley and Sons Inc.
U.S.A., 2008. pp: 67-70. [24] S.Raghavan, ―Handbook of Spices, seasonings and
flavoring‖, 2nd edition, CRC Press, USA, 2007. Pp:
142-143 [25] Gracelin, D.H., Britto, A.J and Kumar,
B.J.R, ―Qualitativeandquantitativeanalysisof
Phytochemicals in five Pteris species.
International
Journal of Pharmacy and Pharmaceutical
Sciences‖,
2012,Vol 5 Sppl. Pp: 105-107
[26] Zuodong Jiang, Chase Kempinski, and Joe
Chappell, ―Extraction and Analysis of Terpenes/Terpenoids‖, 2016 June.
[27] Hsiao JY, Lin ML, ―A Chemotaxonomic study of essential oils from the leaves of genus Clerodendrum (Verbenaceae) native to Taiwan‖, Botany Bulletin Academica Sinica, 1995;36: 247-251.
[28] Essam F. Al-Jumaily and Maytham H. A. Al-Amiry, ―Extraction and Purification of Terpenes from Nutmeg (myristica fragrans)‖, Vol. 15 (3),
September, 2012, pp.151-160. [29] V.A. Parthasarathy, B. Chempakam and T.J.
Zachariah.,
N.K. Leela, ―Nutmeg and mace. In: Chemistry of
Spice. (eds.)‖, UK. Biddles Ltd, King’s Lynn, 2008. Pp: 165-189.
[30] Chung PY, Navaratnam P,and Chung LY,
―Synergistic antimicrobial activity between pentacyclic triterpenoids and antibiotics against
Staphylococcus aureus strains‖, Annals of Clinical Microbiology & Antimicrbioals, 201110:25 doi:10.1186/1476-0711-10-25.
[31] Priti Arya, Savita Pandey, VipinVerma, ―Kidney
stones formation and use of medicinal plants as
anti-urolithiatic agents‖, 2017 July. [32] Trinchieri A, Nespoli R, Ostini F, Rovera F, Zanetti
G,Pisani E. J Urol, ―A study of dietary calcium and
other nutrients in idiopathic renal calcium stone formers with low bone mineral content‖, 1998.
[33] Ghatapanadi SR, Johnson N, Rajasab AH,
―Medicinal plants of north Karnataka used in
treatment of kidney stone and urinary tract
infections‖, The socio scan, 2010; 2(3, 4): 23-24. [34] Xiu-Qiong Y, Jian-Ming O, Hua P, Wen-Yu Z, He-
Qun C, ―Inhibitionon calcium oxalate crystallization and
repair on injured renalepithelial cells of degraded
soybean polysaccharide‖, CarbohydrPolymers,
2012; 90:392—8.
[35] Zhang Y, Tang Y, Xu J, Zhang D, Lu G, Jing
W, ―Modulation of polyepoxysuccinic acid on crystallization of calcium oxalate‖, JSolid State
Chem, 2015;231:7—12.
International Journal of Pure and Applied Mathematics Special Issue
561
International Journal of Pure and Applied Mathematics Special Issue
562
563
564