39

CHAPTER 3

PHYTOCHEMISTRY: ANALYTICAL STUDIES OF

COSTUS IGNEUS (N.E.Br.)

3.1 INTRODUCTION

Phytochemistry evolved from natural products chemistry is confined to the

study of products elaborated by plants and it has developed as a distinct discipline

between natural product organic chemistry and plant biochemistry in recent years.

It deals with the study of chemical structures of plant constituents, their

biosynthesis, metabolism, natural distribution and biological functions (Miller,

1973).

Among the phytochemicals, the polyphenolics constitute a distinct group.

They embrace a wide range of substances, which possess in common an aromatic

ring bearing one or more hydroxy substituent or their ether or glycoside

derivatives. These compounds possess great structural diversity and are of

widespread occurrence among the secondary metabolites. A further feature of this

particular group of compounds is their ability to interact with primary metabolites

such as polysaccharides and proteins. Flavonoid structure is based on that of

flavone (2-phenyl chromone) having basic C6 – C3 - C6 skeleton in common when

the heterocyclic ring is reduced, it becomes flavan (2-phenyl chroman), flavone

consists of two benzene rings (A and B) joined together by a y - ring (ring C). The

various classes of flavonoid compounds differ from one another only by the state

of oxidation of this carbon link (Swain, 1976). There is a limitation to the number

of structures commonly found in nature, which vary in their state of oxidation

from flavan 3-01s (catechin) to flavonols (3- hydroxy flavones) and anthocyanins.

Flavanones, flavanonols or dihydro flavonols and the flavan 3-4-diols

(proanthocyanidins) are also included in the flavonoids. It should be noted that

there are also five classes of compounds (dihydro chalcones or 3-phenyl

40

propiophenones chalcones or phenyl styryl ketones isoflavones or 3-phenyl

chromones neoflavones or 4-phenyl cumarins and the aurones or 2-benzylidine-3-

coumatanones) which do not actually possess the basic 2-phenyl chromone

skeleton, but are closely related both chemically and biosynthetically to other

flavonoid types, that they are always included in the flavonoid group (Swain,

1976).

Steroidal sapogenins are natural products obtained from saponins a group

of glycosides widely distributed in plants (Hostettmann and Marston, 1995). In

nature, most sapogenins have the 22R configuration, and with regard to C-25 there

are two kinds of sapogenins: 25R (the methyl group in C-25 is equatorial

oriented), as in diosgenin and hecogenin or 25S (the methyl group in C-25 is axial

oriented), as in sarsasapogenin. It is very important to highlight the difference of

absolute configuration at this centre because in many side-chain transformations,

products and yields may vary; depending on such configuration.

Costus igneus also known as fiery costus or spiral flag or insulin plant

belonging to the costaceae family, contains a range of phytochemicals viz

flavonoids, alkaloids, terpenoids and it was traditionally used in India to control

diabetes. (Devi and Urooj, 2008; Saraswathi et al., 2010; Bhat, et al., 2010;

Shetty, et al, 2010a; Kripa, et al., 2010; Shetty, et al., 2010). In this study

qualitative, quantitative phytochemical analysis were conducted, in addition to

this an attempt was made to isolation, purification and characterization of

compounds having antidiabetic activity and also to find the fatty compounds from

Costus igneus by GCMS.

3.2 MATERIAL AND METHODS

3.2.1 Cultivation of Costus igneus

The plant was grow in the pot with the potting mix of 1:1:0.2 (red soil:

sand: vermicompost). Regular watering is done sufficient for the plant to grown in

under 50% shade net condition.

41

3.2.2 Extract preparation

3.2.2.1 Solvent extraction

Leaf, stem and rhizome of Costus igneus were collected, cleaned and

shade-dried. The dried each part of Costus igneus were pulverized by a

mechanical grinder and passed through a 20-mesh sieve. A powdered samples

(500g) were separately extracted with petroleum ether, hexane, methanol and

ethanol using a soxhlet apparatus. The extraction was carried out for 24h at room

temperature with mild shaking. The extracts were filtered and concentrated at

35°C and it is used for further analysis (Kameshwara et al., 2003).

3.2.2.2 Preparation of crude sapogenin extract

Freshly harvested leaf and rhizome each weighing 50g were chopped and

refluxed with 3.5M HCl (115ml) for 3h. The solution was filtered, the residue

washed with water to neutrality and the filter and the residue were dried at 65°-

70°C for overnight. The dried residue was then extracted with petroleum ether in a

soxhlet apparatus for 6h and the petroleum ether extract was concentrated. The

resulting solid which precipitated was filtered and dried to give the crude

sapogenin extract (Marie et al., 2005).

3.2.3 Qualitative analysis of phytochemicals in leaf, stem and rhizome of

Costus igneus

Chemical tests were carried out on the aqueous extract and on the

powdered specimens using standard procedures to identify the constituents as

described by Sofowara (1993) and Harborne (1973).

3.2.3.1 Test for tannins

About 0.5g of the powdered samples was boiled in 20ml of water in a test

tube and then filtered. A few drops of 0.1% ferric chloride was added and

observed for brownish green or a blue-black colouration.

42

3.2.3.2 Test for phlobatannins

Deposition of a red precipitate when an aqueous extract of each plant

sample was boiled with 1% aqueous hydrochloric acid was taken as evidence for

the presence of phlobatannins.

3.2.3.3 Test for saponin

About 2g of the powdered sample was boiled in 2ml of distilled water in a

water bath and filtered. 10ml of the filtrate was mixed with 5ml of distilled water

and shaken vigorously for a stable persistent froth. The frothing was mixed with 3

drops of olive oil and shaken vigorously then observed for the formation of

emulsion.

3.2.3.4 Test for flavonoids

Three methods were used to determine the presence of flavonoids in the

plant sample (Sofowara 1993; Harbrone, 1973). 5ml of dilute ammonia solution

were added to a portion of the aqueous filtrate of each plant extract followed by

addition of concentrated Sulphuric acid. A yellow colouration was observed in

extract indicated the presence of flavonoids. The yellow colouration disappeared

on standing.

In the second method, few drops of 1% aluminium solution were added to

a portion of each filtrate. A yellow colouration was observed indicating the

presence of flavonoids. In the third method, a portion of the powdered plant

sample was in each case heated with 10ml of ethyl acetate over a steam bath for

three minutes. The mixture was filtered and 4ml of the filtrate was shaken with

1ml of dilute ammonia solution. A yellow colouration was observed indicating a

positive test for flavonoids.

3.2.3.5 Test for steroids

Two ml of acetic anhydride was added to 0.5g ethanolic extract of each

sample with 2ml sulphuric acid. The colour changed from violet to blue or green

in some samples indicating the presence of steroids.

43

3.2.3.6 Test for terpenoids (Salkowski test)

5ml of each extract was mixed in 2ml of chloroform and concentrated

sulphuric acid (3ml) was carefully added to form a layer. A reddish brown

colouration of the interface was found to show positive results for the presence of

terpenoid.

3.2.3.7 Test for Cardiac Glyosides (Keller-Killani test)

Five ml of each extract was treated with 2ml of glacial acetic acid

containing one drop of ferric chloride solution. It was underlayed with 1ml of

conc. sulphuric acid. A brown ring of the interface indicates a deoxysugar

characteristic of cardenolides. A violet ring may appear below the brown ring

while in the acetic acid layer, greenish ring may form just gradually throughout

thin layer.

3.2.4 Quantitative analysis of phytochemicals in leaf, stem and rhizome of

Costus igneus

3.2.4.1 Preparation of fat free sample

2g of the sample were defatted with 100ml of diethyl ether using a soxhlet

apparatus for 2h.

3.2.4.2 Determination of total phenols by spectrophotometric method

The fat free sample was boiled with 50ml of ether for the extraction of the

phenolic component for 15min. 5ml of the extract was pipetted into a 50ml flask;

10 ml of distilled water was added. 2ml of ammonium hydroxide solution and 5ml

of concentrated amyl alcohol were also added. The samples were made up to mark

and left to react for 30min for colour development. This was measured at 505nm

using spectrophotometer (UV 1800pc, Mapada).

3.2.4.3 Alkaloid determination using Harborne (1973) method

5g of the sample was weighed into 250ml beaker and 200ml of 10%

acetic acid in ethanol was added, covered and allowed to stand for 4h. This was

filtered and the extract was concentrated on a water bath to one-quarter of the

44

original volume. Concentrated ammonium hydroxide was added drop wise to the

extract until the precipitation was complete. The whole solution was allowed to

settle and the precipitated was collected and washed with dilute ammonium

hydroxide and then filtered. The residue was the alkaloid, which was dried and

weighed.

3.2.4.4 Tannin determination by Van-Burden and Robinson (1981) method

500mg of the sample was weighed into a 50ml plastic bottle. 50ml of

distilled water was added and shaken in a mechanical shaker. This was filtered

into a 50ml volumetric flask and made up to the mark. Then 5ml of the filtrate was

pipetted out into a test tube and mixed with 2ml of 0.1M FeCl3 in 0.1N HCl and

0.008M potassium ferrocyanide. The absorbance was measured at 120nm within

10min using spectrophotometer (UV 1800pc, Mapada).

3.2.4.5 Saponin Determination

The samples were ground and 20mg of each was put into a conical flask

and 100cm3

of aqueous ethanol were added. The samples were heated over a water

bath for 4h with continuous stirring at about 55°C. The mixture was filtered and

the residue re-extracted with another 200ml 20% ethanol. The combined extracts

were reduced to 40ml over water bath at about 90°C. The concentrate was

transferred into a 250ml separatory funnel and 20ml of ethyl ether was added and

shaken vigorously. The aqueous layer was recovered while the ether layer was

discarded. The purification process was repeated. 60ml of n-butanol was added.

The combined n-butanol extracts were washed twice with 10ml of aqueous

sodium chloride. The remaining solution was heated in a water bath. After

evaporation the sample were dried in oven to constant weight. The saponin

content was calculated as percentage (Obadoni and Ochuko, 2001).

3.2.4.6 Flavonoid determination

10g of the plant sample was extracted repeatedly with 100ml of 80%

aqueous methanol at room temperature. The whole solution was filtered through

Whatmann filter paper No: 42 (125mm). The filtrate was later transferred into

45

crucible and evaporated into dryness over a water bath and weighed to a constant

weight (Bohm and Kocipai-Abyazan, 1974).

3.2.5 Isolation and characterization of diosgenin and flavonoid compound

3.2.5.1 Isolation of diosgenin

Crude sapogenin extract of Costus igneus was chromatographed on silica

gel column eluting with solvent mixtures increasing the polarity composed of n-

hexane, chloroform, ethyl acetate (ethyl acetate up to 80%) and fractions were

collected. The purity of the all fractions collected was analyzed by thin layer

chromatography on silica gel developed in n-hexane:ethyl acetate (7:3). Spots

were visualized by spaying the plates with 20% antimony chloride in chloroform,

spray reagent. Fraction collected with n-hexane: ethyl acetate (increasing polarity

ethyl acetate up to 80%) were pulled together these fractions were showed single

spot of same Rf value of TLC. It was evaporated in a water bath (50-60°C) to

afford a solid residue. The residue was dissolved in a mixture of CHCl3: EtOH

(40:60) with little warming on a water bath. It was left undisturbed in refrigerator

when needle shaped crystals of diosgenin was obtained.

3.2.5.2 Isolation of flavonoid compound

Crude ethanol extract of Costus igneus was chromatographed on silica gel

column eluting with solvent mixtures increasing the polarity composed of

chloroform, methanol (methanol to 35%) and fractions were collected. The purity

of the all fractions collected was analyzed by thin layer chromatography on silica

gel developed in Toluene: Ethyl acetate: Acetic acid: Methanol (2.5:7:0.25:0.25).

Spots were visualized by spaying the plates with 20% antimony chloride in

chloroform, spray reagent. Fraction collected with n-hexane: ethyl acetate

(increasing polarity methanol up to 35%) were pulled together these fractions

were showed single spot of same Rf value of TLC. It was evaporated in a water

bath (50-60°C) to afford a solid residue. The residue was dissolved in a mixture of

CHCl3:methanol (70:30) with little warming on a water bath. It was left

undisturbed in refrigerator when light yellowish powder of flavonoid was

obtained.

46

3.2.5.3 Characterization of chemical compound

The purified compounds were analysed by GCMS spectrometry in an LKB

model 2091 at ionization energy of 70eV. The 1H and

13C NMR spectra of the

purified diosgenin and flavonoid compounds were recorded at 300MHz and

500MHz, respectively. All the 13

C NMR spectra were recorded in the proton

noise-decoupling mode. Functional group of diosgenin and flavonoid compound

were analysed by FTIR (Arramon et al., 2002).

3.2.6 Isolation and purification of diosgenin and flavonoid compound by High

Performance Liquid Chromatography (HPLC)

3.2.6.1 Sample preparation for HPLC

The crude ethanol and sapogenin extract were evaporated to dryness. The

crystals formed are then dissolved in HPLC grade methanol, filtered with 0.45

micron filter paper from that 15µl was injected (Ganzara et al., 2003).

3.2.6.2 HPLC separation condition

The analytical HPLC system ((Shimadzu, Kyoto, Japan)) was equipped

with a diode array detector at 194nm, a 20µl loop, 200 x 4.6 mm C18 column

(Phenomenex, Nucleosil® 5 µm C18 120 Å LC) methanol (HPLC grade, 0.2mm

filtered) used as a mobile phase. The flow rate was 1.0 ml/min and aliquots of

15µl were injected.

3.2.7 Quantification of flavonoid (quercetin, kaempferol) and sapogenin

(diosgenin, betasitosterol) by HPTLC

3.2.7.1 Sample preparation for HPTLC

The ethanol and sapogenin extracts of Costus igneus leaf, rhizome were

concentrated and followed by acid hydrolysis to liberate aglycones, if any

glycosides were present. About 100mg of extract was weighed and dissolved in

10ml of ethanol from which 5µl was used for quantification of flavonoid

(quercetin, kaempferol) and sapogenin (diosgenin, betasitosterol).

47

3.2.7.2 Chromatography condition

A Camag HPTLC system equipped with an automatic TLC sampler

(ATS4), TLC scanner 3 and integrated software WINCATS version 1.4.1 was

used for the analysis. Chromatography was performed on 20 cm × 10 cm HPTLC

plates coated with silica gel 60 F254 (E.Merck Germany) of 200μm layer

thickness for the quantification of quercetin, kaempferol, diosgenin and

betasitosterol in Costus igneus leaf and rhizome. Standard and samples were

applied to the plates as 8mm long bands, 8mm apart by use of a Camag Linomat

(V) sample applicator equipped with a 100μl microsyringe and an automatic TLC

sampler (ATS4) under a flow of N2 gas.

3.2.7.3 HPTLC method for estimation of flavonoid (quercetin, kaempferol)

and sapogenin (diosgenin, betasitosterol)

3.2.7.3.1 Preparation of calibration curve of standard

One milligram of working standard flavonoid (quercetin, kaempferol) and

sapogenin (diosgenin, betasitosterol) were dissolved in 1ml of methanol and

chloroform respectively to yield stock solution of 100µg/ml concentration.

Calibration curve from 1000-6000ng/spot was prepared and checked for

reproducibility, linearity and validating the proposed method. The correlation

coefficient (r), coefficient of variance (CV%) and the linearity of results were

calculated.

3.2.7.3.2 Method specifications

Silica gel 60 F254 precoated plates (10 x 10 cm) were used with Toluene:

Ethyl acetate: Acetic acid: Methanol (2.5:7:0.25:0.25) as solvent system for

quercetin, kaempferol and hexane: ethyl acetate (7.2:2.5) for diosgenin,

betasitosterol. Sample was spotted on precoated TLC plates by using Linomat 5

spotter. Ascending mode was used for development of thin layer chromatography.

TLC plates were developed up to 10cms. The plates were then sprayed with 20%

antimony chloride in chloroform and dried in a chromatographic oven at 105ºC

for 10min. The contents of flavonoid (quercetin, kaempferol) and sapogenin

(diosgenin, betasitosterol) in the Costus igneus leaf and rhizome were determined

48

by comparing area of the chromatogram of standard of flavonoid (quercetin,

kaempferol) and sapogenin (diosgenin, betasitosterol) respectively (Anjaneyulu

and Chopra, 2004).

3.2.8 Isolation fatty acid compounds in rhizome, stem and leaf ethanolic

extract of Costus igneus by GC-MS (Gas Chromatography with Mass

Spectroscopy)

3.2.8.1 Sample preparation for GC-MS

The rhizome, stem and leaf ethanolic extracts were filtered by using

sodium sulphate in distilled ethanol. Then the sample was collected and injected

into a gas chromatography coupled with a mass spectrometry.

3.2.8.2 GC-MS separation condition

GC Programme: Column: Elite-1(100% Dimethyl polysiloxane),

30×0.25mm ×1µmdf, Equipment: GC Clarus 500 Perkin Elmer, Carrier gas:

1ml per minute, split: 10:1, Detector: Mass detector- Turbo mass gold- Perkin

Elmer, Software: Turbomass 5.2, Sample injected : 2µl. Oven Temperature

programme: 110°C – 2min hold, upto 200°C at the rate of 10°C/min – no hold,

Upto 280°C at the rate of 5°C/min – 9 min hold, Injector temperature: 250°C,

Total GC running time : 36 min, MS programme: Library used: NIST Version-

Year 2005, Inlet line temperature:200°C, Source temperature :200°C, Electron

energy:70 eV, Mass scan (m/z): 45-450, Total MS running time:36min.

3.3 RESULTS

3.3.1Qualitative analysis of phytochemicals in leaf, stem and rhizome of

Costus igneus

Presence of various compounds viz., tannins, phlobatannins, saponin,

flavonoids, terpenoids, cardiac glycosides were analysed in ethanol, methanol,

hexane and petroleum ether extracts of leaf, rhizome and stem of Costus igneus.

All the above mentioned compounds were present in ethanol extract than the

others extracts (Table 3.1).

49

Table 3.1 Qualitative phytochemical analysis of different extract of Costus

igneus leaf, stem and rhizome

1. Tannin, 2. Phlobatannin, 3. Saponin, 4. Flavonoid, 5. Steroid, 6. Terpenoids,

7. Cardiac glycosides

S.No Extracts Plant

parts 1 2 3 4 5 6 7

1 Ethanol

Leaf + + + + + + +

Stem + + + + + + +

Rhizome + + + + + + +

2 Methanol

Leaf + + - + - + +

Stem + + - + - + +

Rhizome + + - + - + +

3 Hexane

Leaf - - + - + - -

Stem - - + - + - -

Rhizome - - + - + - -

4 Petrolium

ether

Leaf - - + - + + +

Stem - - + - + + +

Rhizome - - + - + + +

5 Water

Leaf + + - + - - +

Stem + + - + - - +

Rhizome + + - + - - +

50

Table 3.2 Quantitative phytochemical analysis of Costus igneus leaf, stem and

rhizome

Table 3.3 Solvent ratio for separation of sapogenin and flavonoid compounds

by TLC

S.No Constituents

Amount (mg/kg)

Rhizome Leaf Stem

1 Tannins 2.4 1.9 1.6

2 Phlobatannins 1.6 1.2 1.0

3 Saponin 2.2 1.8 1.0

4 Flavonoids 12 6.9 5.0

5 Steroids 1.4 0.98 0.67

6 Terpenoids 3.0 2.5 1.8

7 Cardiac glycosides 1.7 0.88 0.68

S.

No Compounds Solvents Ratio

Rf

value

1 Sapogenin Hexane : Ethylacetate 7.5:2.5 0.85

2 Flavonoid Methanol : Chloroform 8:2 0.27

51

3.3.2 Quantitative analysis of phytochemicals in leaf, stem and rhizome of

Costus igneus

The rhizome of Costus igneus contains higher amount of tannins,

phlobatannins, saponin, flavonoids, steroids, terpenoids, cardiac glycosides while

compare to leaf and stem as shown in the Tables 3.2 and 3.3.

3.3.3 Isolation and characterization of diosgenin

Diosgenin was isolated and purified from sapogenin extract of Costus

igneus rhizome by repeated silica gel column chromatography followed by

preparative TLC (Rf:0.27) (Figs. 3.11 and 3.1.2). IHNMR

13C NMR

spectroscopies, FTIR and GCMS were used to verify the molecular structure of

the purified steroid sapogenin. The assignments of the signals of this compound in

the spectra were obtained by comparison with the corresponding signals of

diosgenin.

Diosgenin: C27H42O3 (m/z 414 [M]+), white needlelike crystals (petroleum

ether), mp 201-203°C, Rf 0.27, silica gel 60 F254, n-hexane: Ethyl acetate (7:3);

[α]D 20 -123° (c 1.0, CHCl3); IR spectrum (KBr, vmax, cm-1

): diosgenin 3429 cm-

1 (OH), 2974.59 (sp3-H), 1600.36 (C=C), 1361.27 (sp10 H), 1050.74 (C-O), 880,

771, 628; 1HNMR (δ, CDCl3 as solvent, 300 MHz): 0.79 (s, C-18 methyl), 0.79

(d, J = 6.2 Hz; C-27 methyl), 0.79 (J = 7.1 Hz;C-21 methyl), 1.03 (s, C-19

methyl), 3.38 (t,J = 10.6 Hz; C-26a-H), 3.47 (d, dd, J = 10.5 Hz and J approx.

4Hz; C-26,B-H), 3.49 (broad, C-3a-H), 4.41 (q, J=7.lHz; C-16H), 5.35 (broad d,J

= 5.3Hz; C-6H); 13

C NMR (CDCl3, 500 MHz); 37.21 (C1), 31.84 (C2),

71.716(C3), 42.26(C4), 140.795(C5), 121.42(C6), 31.37(C7), 31.43(C8),

50.04(C9), 36.64(C10), 20.87(C11), 39.78(C12), 41.6(C13), 56.51(C14),

31.61(C15), 80.819(C16), 62.84(C17), 16.29(C18), 19.42(C19), 41.6(C20),

14.53(C21), 109.297(C22), 31.35(C23), 28.79(C24), 30.29(C25), 66.84(C26),

17.14(C27; ESI MS, m/z 414 (MI), 398, 355, 342, 327, 300, 282, 271 and 126

(Table 3.4) (Fig. 3.2.1)

52

3.3.4 Isolation, purification and characterization of flavonoid compound

Flavonoid compound was isolated and purified from ethanol extract of

Costus igneus rhizome by repeated silica gel column chromatography followed

by preparative TLC (Rf 0.72) (Fig .3.1.1). 1H NMR spectrum of isolated quecertin

derivative is 6.24, 6.4 (Phenolic carbons attached with flavones ring), 6.8, 7.8, 7.9

(side chain phenyl ring), 3.5(methoxy), 1.0-2.5(alcohol protons). 13

C Spectrum of

isolated quecertin derivative is 163, 161, 147, 144, 146, 135, 114 (Phenolic

carbons attached with flavones ring), 177 (carbonyl carbon), 122, 120, 114, 97

(side chain phenyl ring) (Fig. 3.2.2).

The mass spectrum of purified compound contained signals of m/z 995.38

(MI), 398, 355, 342, 327, 300, 282, 271 and 126.

3.3.5 HPTLC method for estimation of flavonoid (quercetin, kaempferol) and

sapogenin (diosgenin, betasitosterol)

Standard quecertin, kaempferol, diosgenin and betasitosterol showed

single peak in HPTLC chromatogram. The calibration curve of quecertin,

kaempferol, diosgenin and betasitosterol were obtained by spotting standard such

as quecertin, kaempferol, diosgenin and betasitosterol on HPTLC plate

respectively. After development of the plates, they were scanned at 254 nm. The

calibration curve were prepared by plotting the concentration of quecertin,

kaempferol, diosgenin and betasitosterol versus average area of the peak. Costus

igneus leaf and rhizome were analysed by the proposed method. The amount of

quecertin, kaempferol, diosgenin and betasitosterol were computed from

calibration curve as shown in (Figs. 3.3.1, 3.3.2, 3.3.3 and 3.3.4). Data from Table

3.5 revealed that CiREE contained highest quantity of the quercetin (0.794%),

kaempferol (4.2%) and SECiR contained highest quantity of diosgenin (0.5%) and

betasitosterol (3.5%) while CiLEE, SECiL contains 0.692%, 3.1%, 0.34% and

1.334% respectively (Table 3.5).

53

Fig. 3.1.1 Isolation of sapogenin and flavonoid by TLC, a) Isolation of saponin,

b) Isolation of flavonoid, c) Flavonoid conformation test.

Fig. 3.1.2 Diosgenin purity analysis by HPTLC

a) b) c)

54

Fig. 3.2.1 a) H1

NMR spectrum of diosgenin, b) Enlarge of H1

NMR spectrum

a)

b)

55

Fig. 3.2.1 c) 13

C NMR spectrum of diosgenin, d) GCMS analysis of diosgenin

c)

d)

56

Table 3.4 Diosgenin 13

C NMR spectrum

Carbon

Number

13

C NMR Carbon

Number

13

C NMR

1 37.21 15 31.61

2 31.84 16 80.819

3 71.716 17 62.84

4 42.26 18 16.29

5 140.795 19 19.42

6 121.42 20 41.6

7 31.37 21 14.53

8 31.43 22 109.297

9 50.04 23 31.35

10 36.64 24 28.79

11 20.87 25 30.29

12 39.78 26 66.84

13 41.6 27 17.14

14 56.51

57

Fig. 3.2.2 a) H1

NMR spectrum of quercetin derivative, b) Enlarge of H1

NMR

spectrum

a)

b)

58

Fig. 3.2.2 c) 13

C NMR spectrum of quercetin derivative

c)

59

3.3.5.1 Validation of HPTLC method

Linearity: A representative calibration curve of quecertin, kaempferol, diosgenin

and betasitosterol were obtained by plotting peak area of quecertin (1000-

6000ng/spot), kaempferol (100-1200ng/spot), diosgenin (100-600ng/spot) and

betasitosterol (50-500ng/spot) respectively. The correlation coefficient of all

compounds as follows: quercetin -0.9635, diosgenin-0.99781, betasitosterol-

0.9998, kaempferol-0.99957.

Recovery (%): The % recovery of standards as follows: quercetin - 96-98%,

kaempferol-93.5%, diosgenin-94.12%, betasitosterol-95.0%.

Specificity: The spectrum of standard quercetin, kaempferol, diosgenin,

betasitosterol spot and quercetin, kaempferol, diosgenin, betasitosterol in Costus

igneus leaf and rhizome were found to be similar or overlap respectively.

3.3.6 Isolation and purification of diosgenin and flavonoid compound by High

Performance Liquid Chromatography (HPLC)

Fig. 3.4.1 shows the analytical HPLC chromatogram for the isolation of

diosgenin at 0.66 Retention Time (RT) compared with the standard diosgenin

(0.66 RT) at 194nm. Figs. 3.4.2 and 3.4.3 were shows the analytical and

preparative HPLC chromatogram for the isolation of quercetin at 2.5 Retention

Time (RT) compared with the standard quercetin (2.5 RT) at 194 nm. Red

compound was isolated from Costus igneus leaf by preparative HPLC at 5.8 RT

(254 nm) (Fig. 3.4.4).

3.3.7 Isolation fatty acid compounds in rhizome, stem and leaf ethanolic

extract of Costus igneus by GC-MS (Gas Chromatography with Mass

Spectroscopy)

The retention time, molecular weight and name of the compounds were

analysed for the root, stem and leaf of Costus igneus using GC-MS (Fig. 3.5). In

rhizome 11, 3 and 15 compounds were isolated in rhizome, stem and leaf

respectively. The compound names are given in the Table 3.6, 3.7 and 3.8.

60

Fig. 3.3.1 a) Structure of diosgenin, b) HPTLC plate for diosgenin isolation, c)

linearity curve, d) HPTLC chromatogram of standard diosgenin, e) & f) HPTLC

chromatogram for isolation of diosgenin in rhizome and leaf respectively

a)

b)

c)

a)

e)

f)

d)

61

Fig. 3.3.2 a) Structure of Quercetin, b) HPTLC plate for Quercetin isolation, c)

linearity curve, d) HPTLC chromatogram of standard Quercetin, e & f) HPTLC

chromatogram for isolation of Quercetin in rhizome and leaf respectively

a) d)

b)

c) f)

e)

62

Fig. 3.3.3 a) Structure of Kaempferol, b)HPTLC plate for Kaempferol isolation, c)

linearity curve, d) HPTLC chromatogram of standard Kaempferol, e & f) HPTLC

chromatogram for isolation of Kaempferol in rhizome and leaf respectively

a)

c) f)

b) e)

d)

63

Fig. 3.3.4 a) Structure of Betasitosterol, b)HPTLC plate for Betasitosterol

isolation, c) linearity curve, d) HPTLC chromatogram of standard Betasitosterol,

e & f) HPTLC chromatogram for isolation of Betasitosterol in rhizome and leaf

respectively

a)

f) c)

b) e)

d)

64

Table 3.5 HPTLC analysis of Quecertin, Kaempferol, Diosgenin and

Betasitosterol

S.No Compound Rf Correlation

& SD

Recovery

(%)

Amount present

(%)

Rhizome leaf

1

Quecertin in

CiREE

0.72

0.9635, 6.86

96-98

0.794

0.692

2 Kaempferol

in CiREE 0.42 0.99957, 1.35 93.5 4.2 3.1

3 Diosgenin in

SECiR 0.27 0.99781, 1.57 94.12 0.5 0.34

4 Betasitosterol

in SECiR 0.09 0.9998, 1.35 95.0 3.5 1.334

65

Fig. 3.4.1 a) HPLC chromatogram of standard diosgenin, b) HPLC chromatogram

for isolation of diosgenin in rhizome, c) HPLC chromatogram for isolation of

diosgenin in leaf

a)

b)

c)

c)

66

Fig. 3.4.2 a) HPLC chromatogram of standard quercetin, b) HPLC chromatogram

for isolation of quercetin in rhizome, c) HPLC chromatogram for isolation of

quercetin in leaf

a)

b)

c)

67

Fig. 3.4.3 Preparative HPLC chromatogram for isolation of quercetin in rhizome,

b) HPLC chromatogram for isolation of quercetin in leaf, c) HPLC chromatogram

of standard quercetin

a)

c)

b)

68

Fig. 3.4.4 a) TLC separation of red compound, b) Preparative HPLC

chromatogram of red compound

a) b)

69

Fig. 3.5 a) GCMS chromatogram for isolation of fatty compounds in Costus

igneus leaf, b) GCMS chromatogram for isolation of fatty compounds in Costus

igneus rhizome, c) GCMS chromatogram for isolation of fatty compounds in

Costus igneus stem

c)

b)

a)

70

Table 3.6 GCMS analysis of fatty acid in Costus igneus leaf extract

S.No RT

(min) Name of the compound

Molecular

formula MW

Peak

area

(%)

1 10.30 Dodecanoic acid C12H24O2 200 2.88

2 14.35 Tetradecanoic acid C14H28O2 228 5.22

3 16.05 Pentadecanoic acid C15H30O2 242 1.09

4 16.77 Hexadecanoic acid, methyl ester C17H34O2 270 0.15

5 17.65 Hexadecenoic acid, Z-11 C16H32O2 254 1.67

6 18.87 n-Hexadecanoic acid C16H32O2 256 19.53

7 19.85 9,12-Octadecadienoic acid (Z,Z) C18H32O2 280 0.86

8 20.00 9octadecanoic acid (Z) methyl ester C19H36O2 296 0.46

9 21.26 9,12-Octadecanoic acid, ethyl ester C20H36O2 308 6.58

10 21.39 Ethyl oleate C20H38O2 310 4.13

11 22.42 Oleic acid C18H34O2 282 21.60

12 22.73 Octadecanoic acid C18H36O2 284 6.48

13 25.66 Hexanedioic acid, bis (2-ethylhexyl)

ester C22H42O4 370 2.94

14 28.78 1,2-Benzenedicarboxylic acid,

diisooctyl ester C24H38O4 390 18.44

15 34.89 Squalene C24H38O4 410 8.02

71

Table 3.7 GCMS analysis of fatty acid in Costus igneus rhizome extract

S.No RT

(min) Name of the compound

Molecular

formula MW

Peak

area

(%)

1 11.6 Dodecanoic acid C12H24O2 200 1.80

2 14.29 Tetradecanoic acid C14H28O2 228 1.48

3 15.76 Pentadecanoic acid C15H30O2 242 0.74

4 17.01 Hexadecenoic acid, Z-11 C16H32O2 254 0.88

5 17.31 n-Hexadecanoic acid C16H32O2 256 12.70

6 17.67 Hexanedioic acid, ethyl ester C18H36O2 284 1.40

7 20.02 9,12-Octadecadienoic acid (Z,Z) C18H32O2 280 6.84

8 20.11 Oleic acid C18H34O2 282 16.63

9 20.31 9,12-Octadecanoic acid, methyl

ester

C19H34O2 308 1.75

10 20.44 Octadecanoic acid C18H36O2 284 5.58

11 26.21 1,2-Benzenedicarboxylic acid,

diisooctyl ester

C24H38O4 390 49.61

Table 3.8 GCMS analysis of fatty acid in Costus igneus stem extract

S.No RT

(min)

Name of the compound Molecular

formula

MW Peak

area

(%)

1 16.16 n-hexadecanoic acid C16

H32

O2 256 35.29

2 18.81 1,6-Octadiene,5,7-dimethyl-

,(R)-

C10

H18

138 11.76

3 24.67 Di-n-octyl phthalate C24

H38

O4 390 52.94

72

4.0 DISCUSSION

The fresh leaf, rhizome and stem of Costus igneus were extracted with

different solvent such as petroleum ether, hexane, ethanol, methanol and water.

Among the different extracts, ethanol extract contained most of the compounds

such as Tannins, Phlobatannins, Saponin, Flavonoids, Terpenoids and Cardiac

Glycosides. Quantitative phytochemical analysis revealed that Saponin and

Flavonoids were high in rhizome than leaf and stem.

Present study focused on isolation, purification and characterization of

phytochemical components of Costus igneus. Earlier study reported that flavonoid

diglycoside and sapogenin were studied to responsible for antidiabetic activity in

Costus (family: Costaceae) based on this literature flavonoid and sapogenin

compounds were isolated, purified, quantified and characterized. HPTLC analysis

revealed that sapogenin extract consist of diosgenin, betasitosterol and other

sterols are higher in rhizome of Costus igneus than leaf and stem. Similarly the

concentration of flavonoids such as quecertin and kaempferol higher in rhizome,

when compared to leaf and stem.

In this study high contents of saponins, and flavonoids were observed in

the rhizome of the plant. These phytochemicals exhibit various pharmacological

and biochemical actions when ingested by animals. Saponin is a known anti-

nutritional factor that can reduce the uptake of certain nutrients including

cholesterol and glucose at the gut through intra lumenal physicochemical

interaction or other yet unidentified activity (Price et al, 1987). Steroidal saponins

and alkaloids such as ergot alkaloids have been reported to elicit uterine muscle

activity (Gwotmut and Nwafor, 2001). The content of these phytochemicals may

be associated with the reported oxytocic and abortifacient activity of the plant’s

leaf extract (Offiah and Anynwu, 1989). The plant leaves, rhizome and stem also

contain flavonoids, which are phenolic compounds that serve as flavouring

ingredients of spices and vegetables (Enwere, 1998). Flavonoids and other

phenolic derivatives have been identified in Costus igneus leaves and rhizome.

The chemical constituents of essential oils of stems, leaves and rhizomes

of Costus igneus were identified by GC-MS. The major constituents identified in

73

the leaf of Costus igneus were hexadecanoic acid (19.53%), 9,12-

Octadecadienoic acid, ethyl ester (6.58%), Tetradecanoic acid (5.22%), Ethyl

Oleate (4.13%), Oleic acid (21.60%), Octadecanoic acid (6.48%),

2-benzenedicarboxylic acid, diisoooctyl ester (18.44%), and Squalene (8.02%).

The rhizome contained hexadecanoic acid (12.70%), 9,12- Octadecadienoic acid,

ethyl ester (6.84%), Tetradecanoic acid (1.48%), Oleic acid (16.63%),

Octadecanoic acid (5.58%) and 1,2 benzenedicarboxylic acid, diisoooctyl ester

(49.61%) as important constituents; whereas n-hexadecanoic acid (35.29%), 1,6-

Octadiene 5,7 acid (11.7%) and Di-n-octyl phthalate (52.94%) were the main

compounds in the stem. The essential oils of stems, leaves and rhizomes of the

Costus igneus contained hexadecanoic acid (palmitic acid) as the major

constituent. Palmitic acid cause high LDL (Low Density Lipoprotein) cholesterol

and increase the risk for coronary heart diseases. Consumption of foods rich in

saturated fatty acids and cholesterol has long been recognized as an important

precursor for the development of coronary heart disease and saturated fatty acids

elevate LDL concentration in the blood. Moreover palmitic acid induces

degeneration of myofibrils in rat adult cardiomyocytes. Exposure of adult rat

cardiomyocytes to palmitic acid for 18 h destroyed both the contractile elements

and the cytoskeleton. The deleterious effect of palmitic acid on myofibrils

preceded DNA fragmentation and was detectable 3h after administration of

palmitic acid. In diabetic type 2 a series of studies (Maedler, et al., 2001; Maedler,

2002) report on the roles of high glucose concentrations and different free fatty

acids (FFAs) on b-cell proliferation, apoptosis, function in cultured human islets,

insulin secretion and insulin resistance.

The saturated fatty acid inhibited β-cell growth, impaired insulin secretion

and induced cell death by apoptosis. Disturbances in glucose metabolism have

been implicated in Palmidic acid-mediated β-cell dysfunction and death via the

so-called randle cycle effect, where increased fatty acid oxidation caused a

reciprocal decrease in glucose oxidation (McGarry, 1998). Since glucose

oxidation has been critically linked to the insulin-secreting potency of the hexose,

impairment of glucose-stimulated insulin secretion would result. Indeed, Palmidic

acid-induced reductions in glucose oxidation and utilization have been reported in

74

association with decreases in ATP, insulin content, insulin secretion and the up-

regulation of apoptotic genes.

In conclusion antidiabetic compounds such as qucertine and kaempferol

are flavonoid compounds and diosgenin, beta-sitostreol are sapogenin compounds

were isolated in higher concentration in rhizome when compared to the leaf.

Hexadacanoic acid and other fatty acids are present in higher concentration in

leaf, which leads to enhance the heart problem and beta cell apoptosis. Based on

this phytochemical analysis, rhizome of Costus igneus was taken for antidiabetic

activity study in streptatozocine induced rats.