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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

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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 (2-

methylbutadiene) 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, β-

carotene, α-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

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carried out. Lupeol and Stigmasterol were confirmed by Fourier Transform Infrared

(FTIR), 1H NMR and

13C 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 (37°C) 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 -20°C (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

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(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 45ºC.

The dried extracts were separately redissolved in 1ml of ethanol and sample of

varying concentration (1-3 µl) for Lupeol and (5-30 µl) 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 ATS4,

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 µm-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 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 for 30 minutes, at room temperature (25ºC±2ºC) and

50±5% 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 p-

anisaldehyde in 50 ml glacial acetic acid and 1 ml of 97% H2SO4 and heat at 105ºC)

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

with 5 ml of 97% H2SO4 at 4ºC). 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 µm/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 13

C NMR technique for the confirmation of purity of the compounds.

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6.3.4.3 Precision

A stock solution containing Lupeol and Stigmasterol compounds were

prepared in chloroform and six 10 μl (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 inter-

day 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

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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 100°C 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 μl of

isolated compounds (Lupeol and Stigmasterol) were applied to silica gel plates,

Merck (Germany) 20×20 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 100°C for

10 minutes. Chromatograms were then examined under daylight within 10 minutes.

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6.3.6.2 High-performance liquid chromatography (HPLC)

The analytical HPLC system (Shimadzu) was equipped with a diode array

detector, a 20 µl 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 μl 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;

13C, 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 pre-

coated 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

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identified and isolated by HPTLC techniques. Previous study has reported that

quantitative analysis of Stigmasterol and dl -α-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).

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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 Amount of

compound present

in the plant material

(mean, µg/100 mg)

Amount of

standard

added (µg)

Amount of

standard

found in

mixture (µg)

Recovery (%)

Lupeol 473 473

946

948.00

1420.66

100.21 ± 0.87

100.12 ± 0.44

Stigmasterol 1913 1913

3826

3830.33

5726.00

100.11 ± 1.14

99.77 ± 0.93

Table 6.2 Amount of Lupeol and Stigmasterol in Costus igneus stems

Compound Quantity (mean)

(mg/100 mg)

Mean ± SE CV (% )

Lupeol 0.473 0.473 ± 0.004 0.84

Stigmasterol 1.913 1.913 ± 0.005 0.26

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

Compound Amount

(ng/spot)

Intra-day precision Inter-day precision

Mean area SD %RSD Mean area SD %RSD

Lupeol 1000 2486.54 1.87 0.07 2491.37 3.53 0.14

2000 4903.46 2.85 0.05 4909.63 5.91 0.12

3000 7344.42 1.16 0.01 7340.09 4.96 0.06

Stigmasterol 1000 1583.83 1.53 0.09 1548.55 1.66 0.10

2000 3162.38 1.71 0.05 3214.36 1.98 0.06

3000 4796.25 1.48 0.03 4680.81 1.84 0.03

Table 6.4 Summary of Validation parameter

Parameters Lupeol Stigmasterol

Linearity

(i) Range

(ii) Correlation coefficient

(a) Height

(b) Area

(iii) Rf value

5000-10000 ng

0.99794

0.99914

0.55

1000-6000 ng

0.99956

0.99999

0.58

Precision (%RSD)

(i) Instrument precision (CV%, n=6)

(ii) Method precision (CV%, n=6)

1.33

2.43

1.68

2.94

LOD (ng/spot) 131 80

LOQ (ng/spot) 430 212

Specificity Specific Specific

Robustness Robust Robust

Ruggedness (%RSD) 0.9416 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).

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Table 6.5 Robustness of the method

Compound Amount

(ng/spot)

Mobile phase %RSD

Lupeol 1000 n-Hexane: Ethyl acetate (80:20 v/v)

n-Hexane: Ethyl acetate (70:30 v/v)

0.94

1.42

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)

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 213°C 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

(C≡C 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 (H-

21a, 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 (H-

23, 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 13

C NMR spectrum of Lupeol showed δC: δ 37.17 (C-1), δ

20.9 (C-2), δ 79.0 (C-3), δ 38.0 (C-4), δ 55.2 (C-5), δ 18.01 (C-6), δ 27.9 (C-7), δ

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38.87 (C-8), δ 50.4 (C-9), δ 34.2 (C-10), δ 19.31 (C-11), δ 20.9 (C-12), δ 35.5 (C-13),

δ 40.01 (C-14), δ 25.1 (C-15), δ 29.8 (C-16), δ 40.8 (C-17), δ 48.2 (C-18), δ 48 (C-

19), δ 151.01 (C-20), δ 27.4 (C-21), δ 38.7 (C-22), δ 25.1 (C-23), δ 15.3 (C-24), δ

15.98 (C-25), δ 15.98 (C-26), δ 14.5 (C-27), δ 16.13 (C-28), δ 109.34 (C-29) and δ

18.32 (C-30) (Enamul et al., 2008) (Figure 6.11).

Stigmasterol melting point 165°C 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: δ 5.27-5.12 (d, m(1H,

Vinylic proton), δ 4.9 d, (J=8 Hz) and 5.0 d (J=7 Hz) 2H, broad olefinic proton), δ

3.45 (m, 1H, CHOH), δ 1.14 to 2.21 (m, 18H, 9 X CH2 and 8H, CH proton), δ 0.62

to 1.09 (m, 18H, 6 XCH3) (Figure 6.13). In the 13

C NMR spectrum of Stigmasterol

showed δC: δ 140.75 (C-5), δ 138 (C-6), δ 129 (C-20), δ 121 (C-21), δ 77.45 (C-3), δ

56.8 (C-14), δ 55.9 (C-17), δ 50.1 (C-9), δ 42.3 (C-20), δ 40.5 (C-12), δ 39.6 (C-13),

δ 37.2 (C-4), δ 36.5 (C-1), δ 36.5 (C-10), δ 31.9 (C-8), δ 31.6 (C-22), δ 31.6 (C-7), δ

28.9 (C-16), δ 28.9 (C-25), δ 25.4 (C-16), δ 24.3 (C-15), δ 21.2 (C-28), δ 21.1 (C-

11,26), δ 21.0 (C-27), δ 19.4 (C-19), δ 18.9 (C-21), δ 12.2 (C-18), δ 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

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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

13C 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.

111

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.

112

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

113

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.

114

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

115

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

Lupeol quantified from Costus igneus stems (pink colour).

116

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

Stigmasterol quantified from Costus igneus stems (blue colour).

117

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

Costus igneus

118

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

119

Figure 6.11 13

C NMR spectra of Lupeol in Costus igneus stems.

120

Figure 6.12 FTIR spectra of isolated compound Stigmasterol from the

stem of Costus igneus

121

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

122

Figure 6.14 13

C NMR spectra of Stigmasterol in Costus igneus stems.

123

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

chromatogram of Lupeol in Costus igneus.

124

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

chromatogram of Stigmasterol in Costus igneus.