In vitro evaluation of anti-urolithiatic properties of Strobilanthes crispus extracted using different solvents

Abstract

Traditionally, Strobilanthes crispus is well-known for the treatment of renal diseases. The aim of the present study was to validate the traditional uses of S. crispus by evaluating its anti-urolithiatic activities in vitro. The inhibitory activity against calcium oxalate (CaOx) via aggregation assay and dissolution using titrimetric method were evaluated. The effects of S. crispus and cystone on slope of nucleation and aggregation as well as CaOx crystal growth were evaluated spectrophotometrically. S. crispus was extracted using n-hexane, ethyl acetate, methanol and water. Methanol (5.92 %) yielded the highest percentage of extract and also showed the highest inhibitory activity against aggregation of CaOx crystals (50.54 ± 2.11 %). Ethyl acetate extract had the most effective dissolution effect on CaOx crystals (52.50 ± 2.50 %). S. crispus significantly (p< 0.05) inhibited the slope of nucleation and aggregation of CaOx crystal and reduced crystal density. The present study validated the traditional uses of S. crispus, which was found to show significant anti-urolithiatic activities. However, further studies are recommended for the isolation and identification of active constituents and their in-vivo analysis.

Full text

Research Journal of Chemistry and Environment_____________________________________Vol. 24 (1) January (2020)
Res. J. Chem. Environ.
117
In vitro evaluation of anti-urolithiatic properties of
Strobilanthes crispus extracted using different solvents
Muhammad Tayyab Gul1,2, Ali Sami Dheyab3, Ekremah Kheun Shaker4, Norhayati Muhammad1,5*
and Aslia Natasha Pauzi1
1. Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia (UTHM), Pagoh Educational Hub, KM 1, Jalan Panchor, 84600
Muar, Johor, MALAYSIA
2. Department of Chemical and Life Sciences, Qurtuba University of Science and IT Peshawar, PAKISTAN
3. Department of Medical Laboratory Techniques, Al Maarif University College, Alanbar, IRAQ
4. Al-Rasheed University College, Medical Laboratory Technique, Hay Al-Hussain, Baghdad, IRAQ
5. Centre of Research for Sustainable Uses of Natural Resources (CoR-SUNR), Universiti Tun Hussein Onn Malaysia (UTHM), Parit Raja, 86400 Batu
Pahat, Johor, MALAYSIA
*norhayatim@uthm.edu.my
Abstract
Traditionally, Strobilanthes crispus is well-known for
the treatment of renal diseases. The aim of the present
study was to validate the traditional uses of S. crispus
by evaluating its anti-urolithiatic activities in vitro.
The inhibitory activity against calcium oxalate (CaOx)
via aggregation assay and dissolution using titrimetric
method were evaluated. The effects of S. crispus and
cystone on slope of nucleation and aggregation as well
as CaOx crystal growth were evaluated
spectrophotometrically. S. crispus was extracted using
n-hexane, ethyl acetate, methanol and water.
Methanol (5.92 %) yielded the highest percentage of
extract and also showed the highest inhibitory activity
against aggregation of CaOx crystals (50.54 ± 2.11 %).
Ethyl acetate extract had the most effective dissolution
effect on CaOx crystals (52.50 ± 2.50 %). S. crispus
significantly (p< 0.05) inhibited the slope of nucleation
and aggregation of CaOx crystal and reduced crystal
density. The present study validated the traditional uses
of S. crispus, which was found to show significant anti-
urolithiatic activities. However, further studies are
recommended for the isolation and identification of
active constituents and their in-vivo analysis.
Keywords: Aggregation, Crystallization, Dissolution,
Inhibition, Strobilanthes crispus, in vitro anti-urolithiatic.
Introduction
Urolithiasis is caused by the accumulation of inorganic salts
(such as calcium, oxalate, phosphorus and ammonia) or
organic salts (such as uric acid)14. Kidney stone formation
has multifactorial causes including diet, genetics and
environment9. Relatively small crystals adhere to the
urothelial lining surface and then increase in size13.
In Malaysia and elsewhere in the world, numerous
treatments have been evaluated for urolithiasis, which recurs
within five years (50%). No recommended treatment can
prevent recurrences21. The currently available clinical
treatment for nephrolithiasis is either expensive or is
associated with side effects. Invasive nephrolithiasis
treatment procedures can cause serious complications and
also impose a financial burden on the health care system18.
In Malaysia, medicinal plants have played a prominent role
in the treatment of various diseases. Plants provide a cheap
source of drugs for the majority of world’s population. Data
from in vitro, in vivo and clinical studies show that
phytotherapeutic agents can be used as an alternative or an
adjunctive therapy for urolithiasis management1.
Strobilanthes crispus is a well-known medicinal herb in
Malaysia locally known as Pecah beling and yellow
strobilanthus in English. This plant belongs to the
Acanthaceae family12. The leaves are boiled and had tea or
mixed with other herbs. It is used as a folk medicine in
Malaysia to treat constipation, diabetes and urolithiasis and
to promote diuresis8. This is because this plant has many
calcium carbonate cystoliths and an infusion is slightly
alkaline. A high content of calcium carbonate makes this
plant's boiled water slightly alkaline; this water can be used
as a diuretic20. This plant's pharmaceutical activity is linked
to its phytochemical content.
In S. crispus, various phytochemical groups and components
have been identified such as flavonoids, phenolic acids,
alkaloids and ester glycosides3. Phenolic acids identified in
the ethanol extracts of dried leaves of S. crispus included p-
hydroxybenzoic acid, p-coumaric acid, caffeic acid, vanillic
acid, gentinic acid, ferulic acid and syringic acid.
Additionally, alkaloids such as caffeine and tannin are also
present17. Environmental conditions have a significant effect
on the phytochemical content and composition24. Therefore,
in the present study, different solvents were used for
extraction and the anti-urolithiatic properties of the various
extracts were evaluated.
Material and Methods
Sample collection: Fresh leaves of Strobilanthes crispus
were purchased from supplier Seri Subah, Agrofarm, Negeri
Sembilan, Malaysia, authenticated by Dr. Ab Rasip Bin Ab
Ghani, Senior Research Officer Forest Research Institute
Malaysia, Kepong 52109 Kuala Lumpur. A plant specimen
was submitted to UTHM herbarium with voucher number
(NYM-03-0008) for future reference. Plant samples were
shade dried (25±3 °C) and then ground.

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Sample preparation: The ground plant samples were kept
at room temperature and in a dry place protected from
moisture. The moisture content of the samples was measured
and maintained consistently4. Cystone was used as a positive
control while distilled water was used as a negative control.
Extraction process: Extraction was performed as described
previously with slight modifications11. The plant samples
were extracted via maceration using non-polar solvents (n-
hexane, ethyl acetate, methanol) and polar solvents (water).
The experiment was carried out in triplicate. The following
equation was used to calculate extraction yield:
Total extract yield, Y (%) =Total mass of extraction
Total mass of sample x 100
Phytochemical analysis of plant samples: Phytochemical
analysis was performed as described previously30. The
concentration of all extracts used in these assays was 1
mg/ml.
Evaluation of anti-urolithiatic properties (In-vitro)
Inhibition activity of plant extracts against calcium
oxalate (CaOx) crystals by aggregation assay: The
aggregation assay was performed following a previously
described method with slight modifications15. In addition,
the rates of inhibition of CaOx aggregation by the extracts
were compared with those of the standard drug, cystone.
CaOx crystal solution was prepared by using 10mM calcium
chloride dihydrate and 1.0mM sodium oxalate containing
200 mM NaCl and 10 mM sodium acetate trihydrate. All
tests were conducted at 37°C at 5.7 pH. For crystallization
of CaOx, 25 ml of CaOx solution was transferred to a beaker
and stirred on a hot plate using a magnetic stirrer.
Then, 1 ml of plant extract (1 mg/ml), cystone (1 mg/ml) or
distilled water was added. The addition of 25 ml of
sodium oxalate solution immediately caused the solution to
become turbid. The turbidity formed was measured in terms
of absorbance at 620 nm using UV-Vis spectrophotometer
(U-3900H HITACHI) continuously for 10 min after the
mixing of the chemicals. In fact, the turbidity of the solution
increased indicating nucleation and then decreased after
some time, which indicates aggregation. This experiment
was performed in triplicate. The percentage inhibition rate
of CaOx aggregation was calculated as follows:27
Inhibition % = [1- (Si/Sc)] x 100
where Sc= slope of aggregation without inhibitor (negative
control) and Si= slope of aggregation in the presence of
inhibitor (positive control/ plant extracts).
Estimation of CaOx by titrimetric method: CaOx (10mg)
and plant extract or cystone (100mg) were weighed, packed
together in a semi permeable membrane and carefully
sutured. Then, extract was allowed to suspend in a conical
flask containing 100ml of 0.1M tris buffer. The conical
flasks were kept at room temperature for 7 to 8 h. The
remaining content in the semipermeable membrane
was transferred into a beaker. Next, 1N sulfuric acid (2ml)
was added and titrated with KMnO4 until a light
pink color appeared27. Consequently, 1 ml of 0.9494 N
KMnO4 was equivalent to 0.1898 mg of calcium.
% dissolved of calcium = [(C-T)/C] x 100
where C = precipitate of CaOx remained in control (mg) and
T = precipitate of CaOx remained when test solution was
used (mg).
Statistical analysis: All the experiments were conducted in
triplicate and the data are presented as mean values and
standard deviation. One-way ANOVA was applied on data
using IBM SPSS Statistics software (Version 20.0, USA)
and the level of significance was kept at p< 0.05.
Results and Discussion
Extraction yield: Extraction process is the step prior to
analyzing phytochemical compounds and anti-urolithiatic
properties of samples. Hence, the effect of solvents and
extraction method was studied in terms of extraction yield as
shown in table 1. The solvents were selected based on their
polarities. Polarity of a solvent plays a considerable role in
the extraction process2.
Extraction with methanol led to highest yield (5.92 %)
followed by ethyl acetate (1.80 %), water (1.10 %) and lastly
n-hexane (0.90 %). This result was similar to that of previous
studies wherein the authors reported methanol to be the best
extraction solvent for Calophyllum inophyllum leaf extract29
and grape pomace22. Similar findings have been observed in
other where n-hexane extract was found to be the least
effective extraction solvent for Arisaema jacquemontii.
Phytochemical components associated with anti-
urolithiatic properties of plant extracts: Phytochemical
screening revealed the presence of alkaloids, steroids,
terpenoids, tannins and saponins in all extracted samples.
The amount of detectable phytochemical in each extract is
summarized in table 2.
In the methanolic extract, steroids and terpenoids were
detected in good amount, whereas in the n-hexane extract,
neither were detected. The number of detectable
phytochemicals in every solvent extract is different. This
might be because solvents of different polarities could
selectively extract different types of phytochemicals6,7,25.
Different types of phytochemicals that are present in each
extract might have some positive contribution to the anti-
urolithiatic effect against CaOx crystals either in terms of
inhibition or dissolution properties. Similar findings have
been observed in another study27 which showed that the anti-
urolithiatic activity of the extract was attributable to the
presence of some bioactive phytochemicals

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Table 1
Percentage yield of herbal plant extracts
Herbal plant
Type of solvent
Mass of sample
(g)
Mass of extract
(g)
Yields (%)
Strobilanthes crispus
n-Hexane
50
0.45
0.90
Ethyl acetate
50
0.91
1.82
Methanol
50
2.96
5.92
Aqueous
50
0.55
1.10
Table 2
The amount of detectable phytochemical of each solvent
Type of solvent
Alkaloid
Steroid
Terpenoid
Tannin
Saponin
n-Hexane
+
-
-
+
-
Ethyl acetate
+
+
+
-
-
Methanol
-
++
++
+
+
Aqueous
-
+
+
+
+
“+” indicates present in trace but detectable amount, “++” indicates phytochemicals in good amount; “–“ absent.
Evaluation of anti-urolithiatic properties (In-vitro)
Inhibitory effect of S. crispus on CaOx crystals: Figure 1
and table 3 show that the methanol extracts of S. crispus
show the highest percentage inhibition of CaOx at 50.54 ±
2.11%. This is due to the presence of various bioactive
compounds including saponins, tannins, terpenoids and
steroids in the extract. Similar types of phytochemicals were
found in the aqueous extract of S. crispus except that the
amount of terpenoid and steroid was quite high. The
difference in inhibition percentage value between methanol
and aqueous extract might be influenced by the amount of
terpenoids and steroids present in the extract. Based on
another study10, the presence of terpenoids was proven to be
useful in the inhibition activity against kidney stone crystals.
Additionally, terpenoids blocked the formation or
precipitation of CaOx, which is similar to the findings of
another study26 that terpenoids have the ability to reduce the
size and area of CaOx formation effectively.
Furthermore, steroids in both extracts also have the potential
to inhibit the rate of CaOx crystallization. This was
confirmed by a previous study on the anti-urolithiatic
activity of the root of Boerhaavia diffusa Linn. which
indicated that steroid could be one of the phytochemicals
that reduces the crystallization of the kidney stone5.
In comparison with methanol and water extract, ethyl acetate
extract showed lower inhibition percentage (23.16±2.11 %),
probably attributable to the low amount of alkaloids,
terpenoids and steroids as compared to that in the other
extracts. Moreover, saponins and tannins are absent in the
ethyl acetate extract. Lastly, the n-hexane extract of S.
crispus possessed the lowest inhibition percentage (14.39 ±
1.6 %). The inhibitory activity of S. crispus extract decreased
with the absence of some phytochemicals such as saponin
and tannin.
Dissolution of CaOx crystals by titrimetric assay: As
shown in figure 2 and table 4, the highest inhibition
percentage (52.50 ± 2.50 %) was noted for the ethyl acetate
extract of S. crispus. This extract with proven anti-
urolithiatic activity was found to contain low amounts of
alkaloids, terpenoids and steroids as secondary metabolites;
however, this amount does not affect the dissolution activity
on CaOx crystals. This was also confirmed in another study19
which reported that crude extracts of Launea procumbens
despite having low amounts of bioactive compounds
potentially dissolved CaOx crystals.
The second highest inhibition percentage was for n-hexane
(45.05 % ± 2.20) followed by aqueous extract (44.50 % ±
1.73) and methanol (36.67 % ± 3.82) extract of S. crispus.
These extracts showed moderate dissolution effects with no
significant difference (p <0.005). Based on phytochemical
screening, aqueous and methanol extracts contained similar
type of phytochemicals including saponins, tannins,
terpenoids and steroids, while the hexane extract only has
alkaloids and tannins.
The difference in percentage between four solvent extracts
of S. crispus for CaOx dissolution might be due to the
presence of alkaloids. Alkaloids help improve the efficiency
of ethyl acetate and hexane extract in the dissolution of
CaOx crystals. Previous studies on Bryophyllum
pinnatumare28, Kalanchoe pinnata, Emblica officinalis,
Bambusa nutans and Cynodon dactylon8 reported that the
medicinal and pharmacological properties including the
anti-urolithiatic properties of this plant were ascribed to the
presence of various phytochemicals including alkaloids.
Similarly, Phaseolus vulgaris seed extract was found to
reduce the size of CaOx monohydrate and dihydrate crystals
which was attributable to the polyphenolics in the extract
including alkaloids23.
Conclusion
The ethyl acetate extract of leaves of S. crispus showed
effective calcium stone inhibition activity as compared to the

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marketed formulation cystone, whereas the methanol extract
was effective against CaOx dissolution. Additional studies
are needed on ethyl acetate and methanol extract of the
leaves of S. crispus to isolate, purify and characterize
bioactive compounds and to identify their possible in-vivo
mechanism of action.
Table 3
Percentage of inhibition of CaOx aggregation by plant extract and the standard drug cystone.
Herbal plant/ Standard drug
Type of solvent
Inhibition percentage (%)
(Mean ±Standard Deviation)
Cystone
-
92.28 ± 0.61a
Strobilanthes crispus
n-Hexane
14.39 ± 1.61e
Ethyl acetate
23.16 ± 2.11d
Methanol
50.54 ± 2.11b
Aqueous
44.83 ± 2.89c
a, b, c,,, Values designated with different alphabets are significantly different from each other.
Table 4
Percentage of dissolution of CaOx crystals by plant extract and the standard drug cystone.
Herbal plant/ Standard drug
Type of solvent
Dissolution percentage (%)
(Mean ±Standard Deviation)
Cystone
-
73.33 ± 3.82a
Strobilanthes crispus
n-Hexane
45.05 ± 2.20g,h
Ethyl acetate
52.50 ± 2.50e,f,g
Methanol
36.67 ± 3.82h
Aqueous
44.50 ± 1.73g,h
a, b, c,,, Values designated with different alphabets are significantly different from each other.
Fig. 1: CaOx inhibition activity of four solvent extracts
of S. crispus
Fig. 2: CaOx disolution activity of four solvent extracts
of S. crispus.
Acknowledgement
The research is funded by the Ministry of Education
Malaysia under Fundamental Research Grant Scheme
(FRGS) Vot No. 1646 and partially sponsored by Universiti
Tun Hussein Onn Malaysia.
References
1. A'attiyyah A., Ghazali Wasw, Ali Nam, Ponnuraj K.T.,
Mohamad S. and Azlina A., Phytochemical Properties and
Traditional Uses of Selected Medicinal Plants in Malaysia: A
Review, Journal of Biomedical and Clinical Sciences, 2, 14-25
(2018)
2. Ahmad I., Sabah A., Anwar Z., Arif A., Arsalan A. and Qadeer
K., Effect of solvent polarity on the extraction of components of
pharmaceutical plastic containers, Pakistan Journal of
Pharmaceutical Sciences, 30(1 Suppl), 247-252 (2017)
3. Al-Henhena N., Khalifa S.A., Ying R.P.Y., Hassandarvish P.,
Rouhollahi E., Al-Wajeeh N.S., Ali H.M., Abdulla M.A. and El-
Seedi H.R., Chemopreventive effects of Strobilanthes crispus leaf
extract on azoxymethane-induced aberrant crypt foci in rat colon,
Scientific Reports, 5, 13312 (2015)
4. Azwanida N., A review on the extraction methods use in
medicinal plants, principle, strength and limitation, Med. Aromat.
Plants, 4, 3-8 (2015)
5. Behera P.C. and Ghosh M., Evaluation of antioxidant,
antimicrobial and antiurolithiatic potential of different solvent
extracts of Aerva lanata linn flowers, Pharmacognosy Magazine,
14(53), 53-57 (2018)
6. Chavan J., Jagtap U., Gaikwad N., Dixit G. and Bapat V., Total
phenolics, flavonoids and antioxidant activity of Saptarangi
(Salacia chinensis L.) fruit pulp, Journal of Plant Biochemistry and
Biotechnology, 22(4), 409-413 (2013)
45.05 52.5
36.67 44.5
0
10
20
30
40
50
60
Hexane
extract Ethyl
acetate Methanol Aqueous
% Dissolution
14.39 23.16
50.54 44.83
0
10
20
30
40
50
60
Hexane
extract Ethyl
acetate
extract
Methanol
extract Aqueous
extract
% Inhibition

Research Journal of Chemistry and Environment_____________________________________Vol. 24 (1) January (2020)
Res. J. Chem. Environ.
121
7. Dailey A. and Vuong Q.V., Effect of extraction solvents on
recovery of bioactive compounds and antioxidant properties from
macadamia (Macadamia tetraphylla) skin waste, Cogent Food and
Agriculture, 1(1), 46-56 (2015)
8. Devbhuti D., Gupta J.K., Devbhuti P. and Bose A.,
Phytochemical and acute toxicity study on Bryophyllum
calycinum SALISB, Acta Poloniae Pharmaceutica-Drug
Research, 65(4), 501-4 (2008)
9. Edvardsson V.O., Ingvarsdottir S.E., Palsson R. and Indridason
O.S., Incidence of kidney stone disease in Icelandic children and
adolescents from 1985 to 2013: results of a nationwide study,
Pediatric Nephrology, 33(8), 1375-84 (2018)
10. Eweka A.O. and Enogieru A., Effects of oral administration of
Phyllanthus amarus leaf extract on the kidneys of adult wistar rats-
a histological study, African Journal of Traditional,
Complementary and Alternative Medicines, 8(7), 307-311 (2011)
11. Fermeglia M., 2 Role of Process Simulation in Extraction
Technologies for Medicinal and Aromatic Plants, Extraction
Technologies for Medicinal and Aromatic Plants, 55-66 (2008)
12. Ghasemzadeh A., Jaafar H.Z. and Rahmat A., Phytochemical
constituents and biological activities of different extracts of
Strobilanthes crispus (L.) Bremek leaves grown in different
locations of Malaysia, BMC Complementary and Alternative
Medicine, 15(1), 422 (2015)
13. Graham T., Complimentary Medicine: A Natural and Healthy
Approach to Stone Prevention, Kidney Stone Disease, 181-185
(2015)
14. Gupta A.K., Dobriyal R. and Victorian T.D., In vitro evaluation
of antiurolithiatic activity of Euphorbia thymifolia L. plant
extracts, International Research in Medical and Health Science,
1(1), 3-7 (2018)
15. Hess B., Jordi S., Zipperle L., Ettinger E. and Giovanoli R.,
Citrate determines calcium oxalate crystallization kinetics and
crystal morphology- studies in the presence of Tamm–Horsfall
protein of a healthy subject and a severely recurrent calcium stone
former, Nephrology Dialysis Transplantation, 15(3), 366-374
(2000)
16. Iqbal M., Bakht J. and Shafi M., Phytochemical screening and
antibacterial activity of different solvent extracted samples of
Arisaema jacquemontii, Pakistan Journal of Pharmaceutical
Sciences, 31(1), 75-81 (2018)
17. Jinfeng E.C., Rafi M.I.M., Hoon K.C., Lian H.K. and Kqueen
C.Y., Analysis of chemical constituents, antimicrobial and
anticancer activities of dichloromethane extracts of
Sordariomycetes sp. endophytic fungi isolated from Strobilanthes
crispus, World Journal of Microbiology and Biotechnology, 33(1),
5 (2017)
18. Kashmar A.M. and Naser E.H., Comparison Between Two
Extraction Methods on Total Extract with Primary Investigation of
Phytochemical Compounds of Some Medicinal Plants used in
Treatment of Urinary Tract Disease, Karbala Journal of
Pharmaceutical Sciences, 13, 29-36 (2017)
19. Niharika M., Himabindu J. and Ramanjaneyulu K., Evaluation
of in vitro antiurolithiatic activity of Tridax procumbens,
International Journal of Scientific Research, 7, 38-39 (2018)
20. Nurraihana H., Norfarizan-Hanoon N., Hasmah A. and Wan
Rosli W., Phytochemical and Antioxidant Potential of Four
Traditional Malaysian Medicinal Plants, J. Trop. Resour. Sustain
Sci, 5, 9-14 (2017)
21. Ordon M. et al, A population based study of the changing
demographics of patients undergoing definitive treatment for
kidney stone disease, The Journal of Urology, 193, 869-874 (2015)
22. Pintać D., Majkić T., Torović L., Orčić D., Beara I., Simin N.,
Mimica–Dukić N. and Lesjak M., Solvent selection for efficient
extraction of bioactive compounds from grape pomace, Industrial
Crops and Products, 111, 379-390 (2018)
23. Prasad K., Antiurolithiatic activity of Phaseolus vulgaris seeds
against ethylene glycol-induced renal calculi in Wistar rats,
International Journal of Green Pharmacy, 11, 281-289 (2018)
24. Gul R., Jan H.U., Gul M.T., Khan M.A., Malik A., Jan S.,
Rafiullah A.S., Dheyab A.S. and Muhammad N., In-vivo
pharmacological investigation of root extract of Myrtus communis
L. by using Animal model (Mus musculus), International Journal
of Botany Studies, 4(2), 7-10 (2019)
25. Rebey I.B. et al, Effects of extraction solvents and provenances
on phenolic contents and antioxidant activities of cumin (Cuminum
cyminum L.) seeds, Food and Bioprocess Technology, 5, 2827-
2836 (2012)
26. Sharifa A., Jamaludin J., Kiong L., Chia L. and Osman K.,
Anti-urolithiatic terpenoid compound from Plantago major Linn.
(Ekor Anjing), Sains Malaysiana, 41, 33-39 (2012)
27. Sharma D., Dey Y.N., Sikarwar I., Sijoria R., Wanjari M.M.
and Jadhav A.D., In vitro study of aqueous leaf extract of
Chenopodium album for inhibition of calcium oxalate and brushite
crystallization, Egyptian Journal of Basic and Applied Sciences, 3,
164-171 (2016)
28. Sohgaura A.K., Bigoniya P. and Shrivastava B., In vitro
antilithiatic potential of Kalanchoe pinnata, Emblica officinalis,
Bambusa nutans and Cynodon dactylon, Journal of Pharmacy and
Bioallied Sciences, 10, 83 (2018)
29. Susanto D., Hapsari S., Trilutfiani Z., Borhet A., Aparamarta
H., Widjaja A. and Gunawan S., Effect of solvent polarity levels
on separation of xanthone and coumarin from Calophyllum
inophyllum leaves extract, Paper presented at the IOP Conference
Series: Materials Science and Engineering (2018)
30. Tiwari P., Kumar B., Kaur M., Kaur G. and Kaur H.,
Phytochemical screening and extraction: a review, Internationale
Pharmaceutica Sciencia, 1, 98-106 (2011).
(Received 22nd January 2019, accepted 28th April 2019)