CHAPTER 6

ISOLATION, PURIFICATION AND CHARACTERIZATION OF LUPEOL

AND STIGMASTEROL FROM THE STEMS OF COSTUS IGNEUS

6.1 INTRODUCTION
Phytochemical evaluation is one of the tools for quality assessment, which
includes preliminary phytochemical screening, chemoprofiling and marker compound
analysis using modern analytical techniques. Use of chromatography for
standardization of plant products was introduced by the WHO and is accepted as a
strategy for identification and evaluation of the quality of plant medicines (Kamlesh
et al., 2010). HPLC and HPTLC both emerged as efficient tool for the phytochemical
evaluation. HPTLC is a widely accepted technique for its high accuracy, precision
and reproducibility of results. In addition, HPTLC has many advantages because of
high sample throughput at low operating cost, easy sample preparation, short analysis
time and analytical assurance (Suthar et al., 2001; Di et al., 2003; Larsen et al.,
2004).
TLC or HPTLC is primarily used as an inexpensive method for separation,
qualitative identification, or semi-quantitative visual

analysis

of

samples.

Accordingly, TLC is often described as a pilot method for HPLC (Rozylo and
Janicka, 1996). However, recent reviews show that the TLC and HPTLC techniques
can be used to solve many qualitative and quantitative analytical problems in a wide
range of fields, including medicine, pharmaceuticals, chemistry, biochemistry, food
analysis, toxicology and environmental analysis (Weins and Hauck, 1996). The use of
TLC/HPTLC has expanded considerably due to the development of forced flow (FF)
and gradient TLC methods, improved stationary and mobile phase selection, as well
as new methods of quantitation methods (Poole and Poole, 1994).
Secondary metabolites are natural products that often have an ecological
role in regulating the interactions between plants and their environment. They can be
defensive substances, such as phytoalexins and phytoanticipins, anti-feedants,
attractants and pheromones (Hanson, 2003). The importance of plant secondary

97

metabolites in medicine, agriculture and industry has led to numerous studies on the
synthesis, biosynthesis and biological activity of these substances (Gershenzon and
Kreis, 1999). The terpenes are biosynthetically constructed from isoprene (2methylbutadiene) units (Ruzicka, 1953). The C5H8 isoprenes polymerise and
subsequently fix the number and position of the double bonds. The basic molecular
formulae of terpenes are thus multiples of C5H8 (Gershenzon and Dudareva, 2007).
Triterpenes comprise a large number of different types of compounds which may be
divided into more important chemical structure families. The main groups of
triterpenoids are represented by pentacyclic derivatives of lupeol (Patocka, 2003).
The 3-O-acyl-derivatives of lupeol have anti-inflammatory properities and many of
them are present in different medicinal plants, as are lupeol acetate and lupeol
docosanoylate in Willughbeia firma (Subhadhirasakul et al., 2000). Steroids are used
in the commercial synthesis of a large number of steroid hormone analogs. A
sapogenin, hecogenin, obtainable in quantity from the waste of sisal plants, is used for
synthesis of cortisol. Stigmasterol, which is readily obtainable from soybean oil, can
be transformed easily to progesterone and to other hormones, and commercial
processes based on this sterol have been developed.
Costus igneus (Costaceae) is traditionally used in India to control diabetes
which is also known as fiery costus or spiral flag or insulin plant are rich in
protein(18%), iron(40mg) and antioxidant components such as ascorbic acid, carotene, -tocopherol, glutathione, phenols, flavonoids, steroids, alkaloids and
terpenoids (Devi and Urooj, 2008; Devi and Urooj, 2010). However, no single
method was found in literature to our knowledge to detect both antiurolithiatic
compounds Lupeol and Stigmasterol in Costus igneus stems.The developed method
was optimized and validated in accordance with International Conference on
Harmonization (ICH) guidelines.
6.2 SPECIFIC AIM
A simple high performance thin layer chromatographic method for the rapid
analysis of Lupeol and Stigmasterol compounds in Costus igneus stems has been

98

carried out. Lupeol and Stigmasterol were confirmed by Fourier Transform Infrared
(FTIR), 1H NMR and

13

C NMR spectra. The method was found suitable for rapid

screening of plant materials for their genotypic assessment and can be performed
without any special sample pretreatment.

6.3 MATERIALS AND METHODS

6.3.1 Collection of plant materials
The medicinal plants Costus igneus (Stem) used in this experiment were
collected from the nursery of the Periyar Maniammai University, Vallam, Thanjavur
and identified at Rapinat herbarium, St. Joseph College, Tiruchirapalli, Tamil Nadu,
India. The voucher of these plants was deposited at the herbarium of the Periyar
Maniammai University, Vallam, Thanjavur. All chemicals and solvents used were of
analytical and HPLC grade.

6.3.2 Preparation of ethanol extracts

The stem of Costus igneus was air-dried at room temperature (37C) for 2
weeks, after which it was grinded to a uniform powder of 40 mesh size. The ethanol
extracts were prepared by soaking 100 g each of the dried powder plant materials in 1
L of ethanol using a soxhlet extractor continuously for 10 hours. The extracts were
filtered through Whatmann filter paper No. 42 (125 mm) to remove all unextractable
matter, including cellular materials and other constitutions that are insoluble in the
extraction solvent. The entire extracts were concentrated to dryness using a rotary
evaporator under reduced pressure. The final dried samples were stored in labelled
sterile bottles and kept at -20C (Hadjzadeh et al., 2007).

6.3.3 Identification and quantification of active compounds from Costus igneus

by HPTLC
6.3.3.1 Sample preparation
All the chemicals, including solvents, were of analytical grade from E. Merck,
India. The HPTLC plates Si 60F254 (20cmX10cm) were purchased from E. Merck

99

(India). Standards of Lupeol (97% purity), Stigmasterol (99% purity) were purchased
from Sigma (New Delhi, India). 100 mg/ml of ethanolic extracts of stem of Costus
igneus was taken for analysis. The extracts were filtered and vacuum dried at 45C.
The dried extracts were separately redissolved in 1ml of ethanol and sample of
varying concentration (1-3 l) for Lupeol and (5-30 l) for Stigmasterol were spotted
for quantification. 1 mg of standard 1 (Lupeol) and Standard 2 (Stigmasterol) were
prepared in 1ml of chloroform, and different amounts of (5000-10000 ng) Lupeol and
(1000-6000 ng) Stigmasterol were loaded onto a TLC plate to get the calibration
curve (Suthar et al., 2001; Badami et al., 2004; Purnima et al., 2007).

6.3.3.2 Thin layer chromatography

A Camag HPTLC system equipped with an automatic TLC sampler ATS 4,
TLC scanner 3 and integrated software Win CATS version 3, was used for the
analysis. Samples were washed on a pre-coated silica gel HPTLC plates Si 60F254
(20cm x 10cm) plate of 200 m-layer thickness, for quantification of Lupeol and
stigmasterol in stem of Costus igneus. The samples and standards were applied on the
plate as 8 mm wide bands with a constant application rate of 150Nl s-1, with an
automatic TLC sampler (ATS4) under a flow of N2 gas, 15 mm from the bottom, 15
mm from the side, and the space between two spots was 6 mm in the plate.

6.3.3.3 Detection and Estimation of Lupeol and Stigmasterol

The linear ascending development was carried out in a Camag twin through
chamber (20cm x 10cm), which was pre-saturated with a 25 ml mobile phase, with nHexane : Ethyl acetate (80:20 v/v) for Lupeol, Toluene: Acetone: Acetic acid (8.9:
0.9 : 0.2 v/v/v) for Stigmasterol for 30 minutes, at room temperature (25C2C) and
505% relative humidity. The length of the chromatogram run was up to 90 mm.
Subsequent to the development; the TLC plate was dried in a current of air, with the
help of air dryer, in a wooden chamber with adequate ventilation. The dried plate was
dipped into freshly prepared Anisaldehyde sulphuric acid reagents (0.5 ml panisaldehyde in 50 ml glacial acetic acid and 1 ml of 97% H2SO4 and heat at 105C)

100

and subsequently in Libermann Burchard reagent (5 ml of acetic anhydride mixed

with 5 ml of 97% H2SO4 at 4C). Quantitative estimation of the plate was performed
in the absorption-reflection mode at 538 nm, using a slit width 6.00 x 0.45 mm, with
data resolution 100 m/step and scanning speed 20 mm/sec. The source of radiation
utilized was a tungsten lamp emitting continuous visible spectra of 366 nm.
Determination of Lupeol and Stigmasterol in extracts of Costus igneus was performed
by the external standard method, using pure standards. Each was carried out in
triplicate.

6.3.4 Method Validation

This method was validated as per the ICH guidelines (International
Conference on Harmonization, 1994, 1996, 2005), the method validation parameters
checked were linearity, precision, accuracy and recovery, limit of detection, limit of
quantification, specificity, Robustness and Ruggedness. All measurements were
performed in triplicates.

6.3.4.1 Calibration Curve and Linearity

The calibration were performed by analysis of working standard solutions of
Lupeol (5000 to 10000 ng for Costus igneus), Stigmasterol (1000 to 6000 ng for
Costus igneus) were spotted on precoated TLC plate, using semiautomatic spotter
under nitrogen stream. The TLC plates were developed, dried by hot air and
photometrically analyzed as described earlier. The calibration curves were prepared
by plotting peak area verus concentration (ng/spot) corresponding to each spot.

6.3.4.2 Recovery
To determine the recovery, known concentrations of standards were added to
a preanalyzed sample of Costus igneus stems. The spiked samples were then analyzed
by the proposed HPTLC method and the analysis was carried out in triplicate.
Quantified Lupeol and Stigmasterol samples were estimated by using FTIR, 1H NMR
and 13C NMR technique for the confirmation of purity of the compounds.

101

6.3.4.3 Precision
A stock solution containing Lupeol and Stigmasterol compounds were
prepared in chloroform and six 10 l (1000 ng /spot) bands were applied and
analyzed by the developed method to determine instrument precision. Six different
volumes of same concentration were spotted on a plate and analyzed by the
developed method to determine variation arising from method itself. To evaluate
intra-day precision, six samples at three different concentrations (1000, 2000 and
3000 ng/ spot) for Lupeol and Stigmasterol were analyzed on the same day. The interday precision was studied by comparing assays performed on three different days.

6.3.4.4 Limit of Detection and Limit of Quantification

The detection limit (LOD) of an individual analytical procedure is the lowest
amount of analyte in a sample which can be detected but not necessarily quantitated
as an exact value. LOD was calculated using the following formula,
3.3 x Standard Deviation of the y-intercept
LOD =

Slope of calibration curve

The quantification limit (LOQ) of an individual analytical procedure is the

lowest amount of analyte in a sample which can be quantitatively determined with

suitable precision and accuracy. LOQ was calculated using the following formula,
10 x Standard Deviation of the y-intercept
LOQ =

Slope of calibration curve

6.3.4.5 Specificity
The specificity of the method was ascertained by analyzing standard
compound Lupeol and Stigmasterol and the compound Lupeol and Stigmasterol is
present in the stem of Costus igneus.

Method Specifications
Silica gel 60 F254 precoated plates (20x 10 cm) were used with n-Hexane:
Ethyl acetate (80:20 v/v) for Lupeol and Toluene : Acetone: Acetic acid (8.9:0.9:0.2

102

v/v/v) for Stigmasterol as solvent system. Sample was spotted on precoated TLC
plates by using Linomat 5 applicator. Ascending mode was used for development of
thin layer chromatography. TLC plates were developing up to 80 mm and scanned in
fluorescence mode at 366 nm. The contents of Lupeol and Stigmasterol in the Costus
igneus were determined by comparing area of the chromatogram of standard Lupeol
and Stigmasterol with calibration curve of the marker compound of Costus igneus,
considering the isolated compound to be 100% pure.

6.3.5 Isolation of Lupeol and Stigmasterol by column chromatography

The condensed ethanol extract of stem powder (1kg) of Costus igneus was
subjected to column chromatography over TLC grade silica gel. Elution of the
column first with petroleum ether, increasing amount of ethyl acetate in petroleum
ether and finally with methanol yielded a number of fractions. The preparation of
solvent systems used to obtain Lupeol (4252mg/898g) and Stigmasterol
(4278mg/224g) were petroleum ether-ethyl acetate (90:10) from fraction 5 and 6. The
compounds were detected on TLC plates by spraying with Libermann Burchard
reagent and heated at 100C for 10 minutes.

6.3.6 Purification of isolated compounds by P-TLC and High performance liquid

chromatography:
6.3.6.1 Preparative Thin-layer chromatography (TLC)
The isolated pure compound was dissolved in appropriate solvents. 5 l of
isolated compounds (Lupeol and Stigmasterol) were applied to silica gel plates,
Merck (Germany) 2020 cm, 0.25 mm in thickness. Plates were developed using the
solvent system n-Hexane : Ethyl acetate (80:20 v/v) for Lupeol, Toluene: Acetone:
Acetic acid (8.9: 0.9 : 0.2 v/v/v)

for Stigmasterol. The separated zones were

visualized with freshly prepared Libermann Burchard reagent and heated at 100C for
10 minutes. Chromatograms were then examined under daylight within 10 minutes.

103

6.3.6.2 High-performance liquid chromatography (HPLC)

The analytical HPLC system (Shimadzu) was equipped with a diode array
detector, a 20 l loop, 200 x 4.6 mm C18 column, methanol: water (HPLC grade, 0.2
mm filtered) used as a mobile phase. The isolated Lupeol and Stigmasterol
compounds were separated using a mobile phase of methanol: water (75:25 v/v) at a
flow rate of 1.0 ml/min, column temperature 30 C. Injection volume was 20 l and
detection was carried out at 254 nm.

6.3.7 Characterization of isolated compounds:

Fourier Transform Infrared (FTIR) spectra were recorded with a nominal
resolution of 4 cm-1 and a wave number range from 400 to 4000 cm-1 using the KBr
pellet technique. 1H and 13C NMR spectra were acquired on Bruker WP 200 SY and
AM 200 SY instruments (1H, 300 MHz;

13

C, 300 MHz) using TMS as internal

standard and CDCL3 as solvent.

6.4 RESULTS AND DISCUSSIONS

6.4.1 Optimization of HPTLC chromatographic conditions
HPTLC fingerprint patterns have been therefore evolved for extracts of Costus
igneus. Lupeol standard was quantitated accurately using silica gel F254 HPTLC precoated plates with the mobile phase for n-Hexane : Ethyl acetate (80:20 v/v), the Rf
value for Lupeol was about 0.55. The chromatographs of standard Lupeol and ethanol
extract of Costus igneus are shown in (Figure 6.1). The Rf value of standard Lupeol
was matched with the Rf value of Costus igneus extract was about 0.55 was shown in
peak (Figure 6.2 (a) and 6.2(b)). Stigmasterol standard was quantitated accurately
using silica gel F254 HPTLC pre-coated plates with the mobile phase Toluene:
Acetone: Acetic acid (8.9: 0.9 : 0.2 v/v/v), the Rf value was about 0.58. The
chromatographs of standard Stigmasterol and ethanol acetate of Costus igneus are
shown in (Figure 6.4). The Rf value of standard Stigmasterol was matched with the
Rf value of extract was about 0.58 was shown in peak (Figure 6.5 (a) and 6.5 (b)). A
pentacyclic triterpenoid compound Lupeol and a steroid compound Stigmasterol were

104

identified and isolated by HPTLC techniques. Previous study has reported that
quantitative analysis of Stigmasterol and dl --tocopherol acetate, two marker
compounds in Leptadenia reticulate by high-performance thin-layer chromatographic
methods (Purnima et al., 2007). Lupeol, a triterpene compound has been isolated
from Crataeva nurvala by HPTLC and also showed antioxaluric and anticalciuric
effects in rats against hydroxyproline-induced hyperoxaluria (Anand et al., 1994b;
Vidya and Varalakshmi, 2000; Suthar et al., 2001; Daudon M and Jungers, 2001;
Enamul et al., 2008). The earlier investigators isolated Lupeol from the methanol
extract of stem bark of Grewia titiaefolia and evaluated the cytotoxic properties on in
vitro cell lines (Touhami et al., 2007).

6.4.2 Validation of HPTLC method

6.4.2.1 Calibration curve and Linearity
The calibration curve was prepared by plotting peak area versus concentration
(ng/spot) corresponding to each spot (Figures 6.3 and 6.6). The regression equation
and correlation curves for Lupeol in Costus igneus were, regression via height
y=149.076+32.745X and r=0.99794, sdv=0.72 (Figure 6.3(a)), regression via area
y=213.109+1731.406X and r=0.99914, sdv=0.72 (Figure 6.3(b)). Stigmasterol in
Costus igneus were, regression via height y= 116.129+0.052X and r= 0.99956, sdv=
1.78 (Figure 6.6 (a)), regression via area y=1732.776+2.151X and r=0.99999,
sdv=0.08 (Figure 6.6(b)).
6.4.2.2 Accuracy and recovery
The results showed that the percentage recoveries after sample processing and
application were in the range of 100.12 % to 100.21 % (Lupeol) and 99.77 % to
100.11 % (Stigmasterol) (Table 6.1). The percentage of Stigmasterol in Costus igneus
stems was higher than that of Lupeol (Table 6.2).

6.4.2.3 Precision
The developed method was found to be precise as indicated by percent RSD
(Relative Standard Deviation) not more than 1.5 (Tables 6.3 and 6.4).

105

6.4.2.4 Specificity
It was observed that the other herbal constituents present in the formulations
did not interfere with the peak of Lupeol and Stigmasterol. Therefore the method was
specific. The spectrum of standard compound Lupeol and Stigmasterol and the
corresponding spot present in Costus igneus matched exactly, indicating no
interference by the other plant constituents and excipients. The peak purity of Lupeol
and Stigmasterol was assessed by comparing the spectra at three different levels like
peak start (S), peak apex (M) and peak end (E) positions of the spot. Good correlation
r = 0.99903 and sdv= 1.37 for Lupeol and r = 0.99977 and sdv= 1.17 for Stigmasterol
were obtained between the standard and sample overlain spectra of Lupeol and
Stigmasterol (Figures 6.7 and 6.8).

Table 6.1 Recovery study of Lupeol and Stigmasterol by HPTLC (n=3)

Compound

Lupeol

Amount of
compound present
in the plant material
(mean, g/100 mg)
473

Stigmasterol

1913

Amount of
standard
added (g)
473
946
1913
3826

Amount of
standard
found in
mixture (g)
948.00
1420.66
3830.33
5726.00

Table 6.2 Amount of Lupeol and Stigmasterol in Costus igneus stems

Compound
Lupeol
Stigmasterol

Quantity (mean)
(mg/100 mg)
0.473
1.913

Mean SE

CV (% )

0.473 0.004
1.913 0.005

0.84
0.26

106

Recovery (%)

100.21 0.87
100.12 0.44
100.11 1.14
99.77 0.93

Table 6.3 Intra-day and inter-day precision of the method (n = 6)

Compound

Amount
(ng/spot)
Lupeol
1000
2000
3000
Stigmasterol 1000
2000
3000

Intra-day precision
Mean area SD
%RSD
2486.54
1.87 0.07
4903.46
2.85 0.05
7344.42
1.16 0.01
1583.83
1.53 0.09
3162.38
1.71 0.05
4796.25
1.48 0.03

Inter-day precision
Mean area SD
%RSD
2491.37
3.53 0.14
4909.63
5.91 0.12
7340.09
4.96 0.06
1548.55
1.66 0.10
3214.36
1.98 0.06
4680.81
1.84 0.03

Table 6.4 Summary of Validation parameter

Parameters
Linearity
(i)
Range
(ii)
Correlation coefficient
(a) Height
(b) Area
(iii)
Rf value
Precision (%RSD)
(i)
Instrument precision (CV%, n=6)
(ii)
Method precision (CV%, n=6)
LOD (ng/spot)
LOQ (ng/spot)
Specificity
Robustness
Ruggedness (%RSD)

Lupeol

Stigmasterol

5000-10000 ng

1000-6000 ng

0.99794
0.99914
0.55

0.99956
0.99999
0.58

1.33
2.43
131
430
Specific
Robust
0.9416

1.68
2.94
80
212
Specific
Robust
0.8114

6.4.2.5 Limit of Detection and Limit of Quantification

The limit of detection was found to be 131 ng/spot for Lupeol and 80 ng/spot
for Stigmasterol while the limit of quantification was found to be 430 ng/spot for
Lupeol and 212 ng/spot for Stigmasterol.

6.4.2.6 Robustness
Robustness tests examine the effect of the operational parameters on the
analysis results. By introducing small changes in mobile phase composition, the
results indicated that the method was robust (Table 6.5).

107

Table 6.5 Robustness of the method

Compound

Amount Mobile phase

(ng/spot)
Lupeol
1000
n-Hexane: Ethyl acetate (80:20 v/v)
n-Hexane: Ethyl acetate (70:30 v/v)
Stigmasterol 1000
Toluene:Acetone:Acetic acid (8.9:0.9:0.2 v/v/v)
Toluene:Acetone:Acetic acid (8.9:1.0:0.1 v/v/v)

%RSD
0.94
1.42
0.96
1.45

6.4.2.7 Ruggedness of the method

It expresses the precision within laboratories variations like different days,
different analyst, and different equipment. Ruggedness of the method was assessed by
spiking the standard 6 times in two different days with different analyst (Table 6.4).

6.4.3 Structural Elucidation of isolated compounds

Lupeol

and

Stigmasterol

were

isolated

by preparative

thin

layer

chromatography of fractions 5-6 from petroleum ether: ethyl acetate (90:10).

Preparative thin layer chromatography was performed in petroleum ether: ethyl
acetate (90:10) on a preparative silica gel plate (Garimella et al., 2001; Barros et al.,
2003; Karadi et al., 2006). Lupeol melting point 213C which corresponds to the
molecular formula C30H50O, IR: (KBr) vmas: 3431.79 cm-1 (Hydrogen bonded OH
Stretch), 2941.78 cm-1 and 2357.89 cm-1 (C-H Stretch in CH2 and CH3), 2103.72 cm-1
(CC Stretch), 1641.94 cm-1 (C=C Symmetric Stretch), 1563.48 (C=C Asymmetric
stretch), 1418.25 cm-1 (C-H deformation in CH2 and CH3), 1365.93 cm-1 (C-H
Stretch), 1036.39 cm-1 (C-O Stretch of secondary alcohol), 887.86 cm-1 (=C-H
bending exocyclic CH2) (Figure 6.9). The 1H NMR: 7.21, 7.19(CDCL3 peak), 4.61,
4.5(H-29, d,d, 2H), 3.14-3.10 (H,3, d,d, 1H, 6 Hz, 5Hz), 2.33(H-19, m, 1H), 2.31 (H21a, m, 1H), 2.12 (H-15A, t, 1H), 2.10 (H-30, s, 3H), 1.61 (H-12A, 1A, d, 2H), 1.44
(H-13, t, 1H), 1.31 (H-2A, d, 1H), 1.24 (H-2B, q, 1H), 1.18 (H-12A, q, 1H), 1.12 (H23, s, 3H), 1.09 (H-15A, d, 1H), 0.97 (H-23,s, 3H), 0.95 (H-27, s, 3H), 0.91 (H-18, t,
6 Hz, 1H), 0.75 (H-28, s, 3H),0.73 (H-24, s, 3H), 0.71 (H-25, s, 3H), 0.68 (H-5, d,
1H) (Figure 6.10). In the 13C NMR spectrum of Lupeol showed C: 37.17 (C-1),
20.9 (C-2), 79.0 (C-3), 38.0 (C-4), 55.2 (C-5), 18.01 (C-6), 27.9 (C-7),

108

38.87 (C-8), 50.4 (C-9), 34.2 (C-10), 19.31 (C-11), 20.9 (C-12), 35.5 (C-13),
40.01 (C-14), 25.1 (C-15), 29.8 (C-16), 40.8 (C-17), 48.2 (C-18), 48 (C19), 151.01 (C-20), 27.4 (C-21), 38.7 (C-22), 25.1 (C-23), 15.3 (C-24),
15.98 (C-25), 15.98 (C-26), 14.5 (C-27), 16.13 (C-28), 109.34 (C-29) and
18.32 (C-30) (Enamul et al., 2008) (Figure 6.11).
Stigmasterol melting point 165C which corresponds to the molecular
formulae C29H48O. IR: 3345.5 cm-1 (br, OH), 2945.9 cm-1 (C-H str. in CH3 and CH2),
1649.8 cm-1 (C=C str.), 1452.6 cm-1 (C-H deformation in gem dimethyl), 1055.8 cm-1
(C-O str. of secondary alcohol) (Figure 6.12). 1H NMR: 5.27-5.12 (d, m(1H,
Vinylic proton), 4.9 d, (J=8 Hz) and 5.0 d (J=7 Hz) 2H, broad olefinic proton),
3.45 (m, 1H, CHOH), 1.14 to 2.21 (m, 18H, 9 X CH2 and 8H, CH proton), 0.62
to 1.09 (m, 18H, 6 XCH3) (Figure 6.13). In the

13

C NMR spectrum of Stigmasterol

showed C: 140.75 (C-5), 138 (C-6), 129 (C-20), 121 (C-21), 77.45 (C-3),
56.8 (C-14), 55.9 (C-17), 50.1 (C-9), 42.3 (C-20), 40.5 (C-12), 39.6 (C-13),
37.2 (C-4), 36.5 (C-1), 36.5 (C-10), 31.9 (C-8), 31.6 (C-22), 31.6 (C-7),
28.9 (C-16), 28.9 (C-25), 25.4 (C-16), 24.3 (C-15), 21.2 (C-28), 21.1 (C11,26), 21.0 (C-27), 19.4 (C-19), 18.9 (C-21), 12.2 (C-18), 12.05 (C-29)
(Figure 6.14). All structures were confirmed by comparison with spectral analysis
data reported in literature (Mohamed Khadeer Ahamed et al., 2007; Jain and Bari,
2010; Kamboj and Saluja, 2011).

6.4.4 Purification of isolated compound by HPLC

The chromatographs of standard Lupeol and the Lupeol isolated from the
ethanol extract of stem of Costus igneus are shown in (Figure 6.15). The Retension
time of Lupeol standard (3.06) was matched with the retension time of Lupeol
isolated from the Costus igneus extract was about (3.04) was shown in peak (Figure
6.15(a) and 6.15(b)). The chromatographs of standard Stigmasterol and the
stigmasterol isolated from the ethanol extract of stem of Costus igneus are shown in
(Figure 6.16). The Retension time of Stigmasterol standard (3.76) was matched with

109

the retension time of Stigmasterol isolated from the Costus igneus extract was about
(3.64) was shown in peak (Figure 6.16(a) and 6.16(b)).

6.5 CONCLUSION
In conclusion, an HPTLC method has been developed with some
modifications and it can be used for the simultaneous quantitative determination of
Lupeol and Stigmasterol in Costus igneus stems; its main advantages are its
simplicity, accuracy, and selectivity. The average recovery values of Lupeol and
Stigmasterol were found to be about 100.16% and 99.94%, which shows the
reliability and suitability of the method. From IR, 1H NMR and

13

C NMR spectral

data, isolated compound was identified as Lupeol and Stigmasterol. Hence, the
assessment of inhibiting effect of aqueous and ethanolic extracts of stem of Costus
igneus and antiurolithiatic compounds Lupeol and Stigmasterol using urolithiasis
induced albino rats under in vivo condition has been carried out in further study.

110

Figure 6.1 Quantitative estimation of Lupeol in Costus igneus.

Figure 6.2 (a) HPTLC chromatogram of standard Lupeol (b) HPTLC chromatogram
of Lupeol in Costus igneus.

111

Figure 6.3 Linear graph for Lupeol in all tracks (concentration vs area)

112

Figure 6.4 Quantitative estimation of Stigmasterol in Costus igneus.

Figure 6.5 (a) HPTLC chromatogram of standard Stigmasterol (b) HPTLC

chromatogram of Stigmasterol in Costus igneus.

113

Figure 6.6 Linear graph for Stigmasterol in all tracks (concentration vs area)

114

Figure 6.7 Spectral comparison of standard Lupeol (green colour) and

Lupeol quantified from Costus igneus stems (pink colour).

115

Figure 6.8 Spectral comparison of standard Stigmasterol (black colour) and

Stigmasterol quantified from Costus igneus stems (blue colour).

116

Figure 6.9 FTIR spectra of isolated compound Lupeol from the stem of
Costus igneus

117

Figure 6.10 1H NMR spectra of Lupeol in Costus igneus stems.

118

Figure 6.11 13C NMR spectra of Lupeol in Costus igneus stems.

119

Figure 6.12 FTIR spectra of isolated compound Stigmasterol from the

stem of Costus igneus

120

Figure 6.13 1H NMR spectra of Stigmasterol in Costus igneus stems.

121

Figure 6.14 13C NMR spectra of Stigmasterol in Costus igneus stems.

122

Figure 6.15 (a) HPLC chromatogram of standard Lupeol (b) HPLC

chromatogram of Lupeol in Costus igneus.

123

Figure 6.16 (a) HPLC chromatogram of standard Stigmasterol (b) HPLC

chromatogram of Stigmasterol in Costus igneus.

124