Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Phytochemical Study of Steroidal Sapogenin “Tigogenin” Present in the Leaves

of Yucca aloifolia Cultivated in Iraq

Nabaa M.Ibrahem
*, 1

and Zainab J. Awad
*

*Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad, Iraq.

Abstract
This study detects the presence of the most important steroidal sapogenin “Tigogenin” in the

leaves of Yucca aloifolia widely cultivated in Iraq. The absence of any study concerning the Tigogenin

content of this medicinal plant in Iraq, and the industrial importance of Tigogenin depending on its role

as a precursor in the synthesis of some steroidal drugs, acquired this study its value. This study

concerned with extraction, identification, isolation, and purification of Tigogenin from the leaves of

Yucca aloifolia. Extraction of this compound was carried out using two methods. Identification of this

compound was done using thin layer chromatography (TLC) where different solvent systems had been

tried. Libermann – Burchard reagent was used for detection. This identification was further augmented

by using high performance liquid chromatography (HPLC) and then this steroidal saponin was isolated

and purified. The identification of isolated Tigogenin was carried out using melting point (M.P.), Thin

layer chromatography (TLC), infrared spectroscopy (IR) and High performance liquid chromatography

(HPLC) . This study confirms the presence of Tigogenin in the leaves of Yucca aloifolia cultivated in

Iraq. Also the result of this study showed that the second extraction method was the best, because the

amount of both extract and Tigogenin were higher than one extraction method.
Key words : Yucca aloifolia , Steroidal saponin , "Tigogenin"

حيائيت للستيرويد الصابوني التيكوجينين الموجود في اوراق نباث اليوكاأدراست كيمو

Yucca aloifoliaالمسروع في العراق
نبأ محمد ابراهيم

،*1
زينب جليل عوادو

، بغذاد ، انعزاق.جايعت بغذاد ،كهٛت انظٛذنت ،انعمالٛز ٔانُباحاث انطبٛت فزع

الخالصة
ْذِ انذراست حخخض بانكشف عٍ ٔجٕد اْى يادة سخٛزٔٚذٚت طابَٕٛت يٕجٕدة فٙ أراق َباث انٕٛكا انًزرٔع فٙ انعزاق ْٔٙ

نهظُاعاث انذٔائٛت يادة انخٛكٕجٍُٛٛ . اٌ عذو ٔجٕد ا٘ دراست فٙ انعزاق حخُأل يحخٕٖ ْذا انُباث يٍ يادة انخٛكٕجٍُٛٛ انًًٓت

بكَٕٓا انًادة االبخذائٛت نخظُٛع بعض االدٔٚت انسخٛزٔٚذٚت انًًٓت يثم انكٕرحزٌٔ ٔانٕٓريَٕاث انجُسٛت ٚبٍٛ لًٛت ْذِ انذراست . فٙ

ًادة ْذِ انذراست حى اسخخالص ٔكشف ٔفظم ٔحُمٛت يادة انخٛكٕجٍُٛٛ انًٕجٕدة فٙ أراق َباث انٕٛكا . حٛث حى اسخخالص ْذِ ان

باسخخذاو طزٚمٍٛ نهفظم ٔحى انكشف عُٓا بٕساطت طزٚمت كزٔياحٕكزافٛا انطبمت انزلٛمت , باسخخذاو يذٚباث يخخهفت كٕسٛظ َالم

بٛزجارد . ٔكذنك بٕساطت حمُٛت كزٔياحٕكزافٛا االداء انعانٙ انسائم ٔبعذْا حًج عًهٛت –ٔانكشف عُٓا باسخخذاو كاشف نٛبزياٌ

ذ اسخخذيج يجًٕعت يٍ انخمُٛاث نهخحمٛك يٍ َٕعٛت انًزكب انًفظٕل ٔدرجت َمأحّ ٔانخٙ شًهج : درجت انفظم ٔانخُمٛت . ٔل

االَظٓار , كزٔياحٕكزافٛا انطبمت انزلٛمت , يطٛاف االشعت ححج انحًزاء ٔكذنك حمُٛت كزٔياحٕكزافٛا االداء انعانٙ انسائم .اثبخج ْذِ

اق َباث انٕٛكا انًزرٔع فٙ انعزاق . كًا اظٓزث ْذِ انذراست باٌ انطزٚمت انثاَٛت انذراست ٔجٕد يادة انخٛكٕجٍُٛٛ فٙ أر

سخخالص . نكٌٕ كًٛت انًسخخهض ٔيحخٕاِ يٍ ْذِ انًادة كاٌ اعهٗ يًااعطخّ انطزٚمت االٔنٗ نال ضمنالسخخالص ْٙ االف
.التيكوجينينمادة ، صابونيتالموادالستيرويديتال،نباث اليوكاالكلماث المفتاحيت :

Introduction

Yucca aloifolia is a shrub or tree – like

plant of the Agavaceae family
(1)

, native to the

deserts of the south – western United States

and northern Mexico
(2)

(Figure 1). This plant

contains several physiologically active

phytochemicals. It is a rich source of steroidal

sapogenins like sarsasapogenin and Tigogenin

and is used commercially as a steroidal

saponin source
(3)

. These sapogenins are

important starting materials for the semi

synthesis of steroidal drugs especially the

cortisone compound and steroidal hormones
(4)

The saponins from Yucca are the main

medicinal agents in the plant that elevate the

body’s production of cortisone possibly the

herb’s ability to aid in arthritic pains
(5)

.Saponins also provide anti – inflammatory

relief as well as the ability to break up

inorganic mineral obstructions and deposis
(6)

1
Corresponding author E-mail:nabaamohammed14@yahoo.com

Received:12/5/2012

Accepted:19/5/2013

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Yucca also has laxative properties and is also

used to establish a flora balance in the GI tract.

It is also speculated that yucca saponin block

release of toxin from the intestines, which

inhibit normal formation of cartilage
(7)

. Both

leaves and roots, function well as diuretic and

emetics and has hair strengthening
(8)

Material and Methods
Plant materials

The plant materials (leaves) of Yucca aloifolia

L. were collected from private garden during

the months of September and October (2010),

they were cleaned and dried in oven at a

temperature between (30 – 40) C for (4 – 5)

hours then these plant materials were coarsely

powdered by mechanical grinder and weighed.

A 50 gm of dried powdered plant materials

were extracted by using two methods.

Extraction

Extraction method No 1
(9) ;

A 50 gm of dried powdered plant

materials (leaves) were extracted in a soxhlet

apparatus with 500ml dichloroethane for 20 –

24 hours. After that the plant material was

dried , then reflexed with 500ml of 4N H2SO4

for 3 hours. After cooling the mixture was

filtered, the residue on the filter was washed

with water, neutralized with 250ml 5% sodium

bicarbonate solution to pH 7.5 and dried at 100

– 105C°. the dry hydrolysate ( the plant

material ) was extracted with petroleum ether

(b.p 60 – 80C°) in soxhlet apparatus for 10

hours. The extract was condensed to small

volume under vacuum and then subjected to

identification (Figure 2).

Figure (1):Yucca alifolia plant

Figure (2): General scheme for method No.1 for extraction of steroidal sapogenin tigogenin from

the leaves of Yucca aloifolia.

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Extraction method No.2
(10)

A 50gm of dried plant material (leaves)

was soaked in water for 24 hours and then

extracted with 80% ethanol (500ml) in soxhlet

extractor for 10 hours. Then the residue was

evaporated to dryness under vacuum. The

dried residue was reflexed with 2N H2SO4 in

water containing 70% 2 – propanol (500ml) at

100C° for 4 hours. The mixture was cooled

and evaporated under vacuum to remove the

alcohol completely, and then the mixture was

neutralized with 5%NH3 then partitioned with

equal volume of petroleum ether (b.p 60 –

80°C) using sepratary funnel to give two

layers, aqueous layer and the petroleum ether

layer. The petroleum ether layer was taken and

evaporated to dryness under vacuum and then

subjected to identification (Figure 3).

Figure (3): General scheme for method No.2 for extraction of steroidal “sapogenin tigogenin”

from the leaves of Yucca aloifolia.

Identification of the steroidal sapogenin

“Tigogenin”:

C.1: Thin layer chromatography (TLC):

In this qualitative identification:- using a

ready-made aluminum plates of silica gel

GF254, one detection method by using

Libermann – Burchard reagen
(11)

comparison with three different developing

solvent systems that were
(12-13)

Solvent 1 (S1):

Chloroform:Methanol (95:5)

Solvent 2 (S2):

Chloroform:Petroleum ether: Methanol

(85:10:5)

Solvent 3 (S3):

Chloroform:Acetone (80:20)

C.2: High performance liquid chromatography

(HPLC):-

Qualitative and quantitative estimations

of Tigogenin component in the crude extract

obtained by extraction methods was carried out

by using high performance liquid

chromatography (HPLC). The identifications

were made by comparism of retention time of

Tigogenin component in the crud extracts with

that of authentic standard at identical

chromatographic conditions. HPLC analysis

was done by using the following conditions:

1) Mobile Phase: Acetonitrile 100%
2) Column: Phenomenex ODS
250mmX4.6mm, 5µm particle size.

3) Column temperature: Ambient
4) Flow rate: 1ml/min
5) Injection volume: 5µL
6) Injection concentration: 1mg/ml

Detection: UV Detector at λ 209nm.

Isolation and purification of Tigogenin:

Isolation and purification of Tigogenin

was done by using the following steps:-
1: Fractionation by column chromatography:

The final residue obtained from extraction

method No2 (best method) was subjected to

column chromatography using glass column

(80cm X 5cm) packed with silica gel (0.063 –

0.200 mm) slurry in (250 ml) CHCl3, in a ratio

of 20gm of silica gel to each 1gm of the

residue. A dry loading of the sample (residue)

was used by dissolving it in small volume of

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Chloroform and adsorbing it on small amount

of silica gel of the same grade used for packing

the column, then dried, grinded and applied to

the top of column in order to prevent clogging.

The column was eluted by gradient elution

technique using CHCl3: methanol with an

increasing percentage of methanol from zero to

100% (the ratios of CHCl3: methanol used

were 100:0, 95: 5, 90:10, 85:15, 80:20, 70:30,

60:40, 50:50, 40:60, 25:75 and CHCl3:

methanol 0:100). The column developed by

adding 50 ml of each eluent with collecting

5ml fractions, then monitored by TLC with

solvent system (S1): Chloroform: Methanol

(95:5). A total number of 100 fractions were

obtained. Those consecutive fractions, which

have the same number of spots with the same

Rf values, were combined and concentrated to

dryness to get major fractions. After that, the

major fractions were subjected to thin layer

chromatography with tigogenin reference

standard and solvent system S1 Chloroform :

Methanol (95:5). the results showed that the

tigogenin compound was found in the major

fraction No.5.

2: Using preparative TLC plates:

Isolation of Tigogenin compound is carried

out using preparative TLC which was

performed by using readymade plates of silica

gel GF254 ( 20 x 20cm ) of 1mm thickness

(Merck). The major fraction (F 5) residue

obtained from column chromatography applied

as a concentrated solution in a row of spots

using capillary tube four times on each plate

(the spots should dry before the next

application). The solvent system used was S1

(Chloroform : Methanol (95:5)) . The detection

was done using Libermann – Burchard reagent

in one side of the plate . The band

corresponding to the tigogenin standared was

scraped out and collected in a beaker, mixed

with chloroform: methanol (95:5), stirred and

left a side for one hour, then filtered. After

evaporation of the solvent, the final filtrate

gives yellowish precipitate .

3.Purification of Tigogenin by using charcoal

material:

The final solid product that has been isolated

by preparative TLC was dissolved by heating

insufficient quantity of methanol and a small

amount of decolorizing charcoal was added,

with stirring to the hot methanol solution until

the supernatant liquid was almost colorless.

Then the hot solution was poured through filter

paper into another flask. Then the solvent was

evaporated to give solid product
(14)

Identification and Characterization of the

Isolated steroidal sapogenin Tigogenin:

1. TLC: Analytical TLC was performed by

using ready made plate. The purified

sapogenin Tigogenin was applied on silica gel

plate as one spot by using capillary tube along

with its standard, using the solvent system

(S1) Chlorform : Methanol (95;5) . The

detection was done by using Liebermann –

burchared reagent .

2. Melting point: The melting point of the
purified sapogenin Tigogenin was done and

compared with that of the available standard

tigogenin.

3. FTIR:Infrared spectra were carried out by
using KBr disc for both purified sapogenin

Tigogenin and its standard.

4. HPLC analysis: HPLC analysis was made
by comparism of retention times obtained at

identical chromatographic conditions of

analyzed purified sapogenin Tigogenin and its

standared. HPLC conditions as mentioned

previously

Results and Discussion:
Extraction methods:

Two methods of extraction of steroidal

sapogenin Tigogenin were tried to select the

best one. Results showed that the method No.2

was better, because the yield of crude extract

was higher than obtained from method No.1.

In addition quantitative estimation by using

HPLC analysis showed that the amount of

Tigogenin obtained by method No.2 was much

more compared with that obtained by method

No.1 as showed in table (1). So, we select

method No. 2 as an extraction procedure in our

work.

Table (1): Quantitative of crud extracts and

Tigogenin obtained from extraction

methods.

Identification of Tigogenin by TLC:

TLC of the crud extracts obtained from the

leaves of Yucca aloifolia by using the

extraction method No.1 and No.2, confirms the

presence of Tigogenin in these extracts in

comparison with tigogenin standard . As

presented in table (2) .and figures ( 4 ,5 and 6).

Extraction

methods

Crud

extract (mg)

Tigogenin

Method No.1 2.9 3.86

Method No. 2 3.5 5

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Table(2): Rf values of Tigogenin from Yucca

aloifolia leaves extract obtained by

extraction methods No.1 and No.2 and its

standard in different developing solvent

systems in TLC

Solvent system S1 S2 S3

Rf value of standard

Tigogenin 0.55 0.74 0.80

Rf value of

Tigogenin in crud

extract of method

No1

0.54 0.75 0.80

Rf value of

Tigogenin in crud

extract of method

No2

0.56 0.78 0.81

S1: Chloroform:Methanol (95:5)

S2: Chloroform:Petroleum ether: Methanol

(85:10:5)

S3: Chloroform:Acetone (80:20)

T E1 E2

Figure(4): TLC for Yucca aloifolia leaves

extract obtained by extraction methods No.1

and No.2 using silica gel GF 254 as

adsorbent and (S1) as solvent system .

Visualization by Liebermann-Burcrhard

spray reagent.

T: Tigogenin standard

E1: extraction method No.1

E2: extraction method No. 2

T E1 E2

Figure (5) : TLC for Yucca aloifolia leaves

extract obtained by extraction methods No.1

and No.2 using silica gel GF 254as

adsorbent and (S2) as solvent system .

Visualization by Liebermann-Burcrhard

spray reagent.

T: Tigogenin standard

E1: extraction method No.1

E1: extraction method No. 2

T E1 E2

Figure (6): TLC for Yucca aloifolia leaves

extract obtained by extraction method No.1

and No.2 using silica gel G 254 as adsorbent

and (S3) as solvent system . Visualization by

Liebermann-Burcrhard spray reagent.

T: Tigogenin standard

E1: extraction method No.1

E2: extraction method No.2

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Isolation and purification of Tigogenin;

One hundred fraction obtained from

column chromatography were monitored by

TLC. The Consecutive fractions that have the

same number of spots of the same Rfvalues

were combined to get 8 major fractions , which

were concentrated to dryness and supjected to

identification , as listed in table ( 3) . In the

first 70 factions , fraction 1-10 gave one spot in

TLC and were collected to give fraction one

(F1) . Fraction (11-20) gave one spot were

collected to give fraction two (F2) , while

fraction 21-30 gave 0ne spot and were

collected to give faction three (F3), fraction

31-40 gave one spot and were collected to give

the fraction four(F4) . Fraction 41-52 gave two

spots and were collected to give the major

fraction five (F5) . Fraction 53- 54 gave one

spot and were collected to give the fraction six

(F6 ) . Fraction 55-57 gave two spots and were

collected to give the fraction seven (F7).

Fraction 58-70 gave the faction eight (F8). In

the last 30 factions no spots were appear in

TLC examination. The results showed that the

tigogenin is found in the fraction five (F5)

therefore we selected this fraction to isolation

thiscompound.

Table (3): Major fractions obtained from

column chromatography

Identification and Characterization of the

isolated Tigogenin

1. Analytical TLC

Isolated compound ( Tigognin) appeared

as a single spot having the Rf value as that of

reference standard .

2. Measuring melting point

The isolated tigogenin was identified

from its sharp melting point of 200 - 202 ºC

compared to standard tigogenin melting point

202-204 ºC .

3. FTIR

The IR spectra of isolated Tigogenin was

gave identical results with that of tigogenin

standard, as showed in table (4) and figures(7

and 8 ) .

Table (4): The characteristic IR absorption bands (in cm-1) of the isolated tigogenin in

comparison with that of tigogenin as reference standard (120)

Major

Fractions

No. of collections

5 ml each

No. of

spots

F1 1-10 1

F2 11-20 1

F3 21-30 1

F4 31-40 1

F5 41-52 2

F6 54-53 1

F7 55-57 2

F8 58-70 4

F9 70-100 Negative

Functional

group

Isolated

tigogenin

Tigogenin

standard
Assignment

Free O-H 3524 3522 Free O-H stretching of alcohol

O-H

Broad band

(3390-3263)

Broad band

(3389-3269)

Broad O-H stretching band indicate hydrogen

bonding

C–H 2929,2848 2941,2874
Asymmetric and symmetric stretching of CH3

and CH2 groups

C-H 1456,1373 1456,1373 C-H bending of CH2 and CH3

C-O

1242-1049

C-O stretching of aliphatic ether

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Figure (7): IR spectrum of tigogenin standard

Figure (8): IR spectrum of isolated and purified tigogenin

4. HPLC analysis

The retention time for the isolated

tigogenin was identical to the main peak of the

standard reference as showed in figures ( 9 and

10 ) .

Figure (9):- HPLC analysis of isolated and purified Tigogenin.

Iraqi J Pharm Sci, Vol.22(2) 2013 Steroidal sapogenin in the leaves of Yucca aloifolia

Figure (10) :- HPLC analysis of Tigogenin standard.

Conclusions:
Phytochemical investigation of Yucca

aloifolia leaves, cultivated in Iraq revealed the

presence of important medicinal natural

product “Tigogenin” belong to steroidal

saponin . Tigogenin was extracted by using

two extraction methods, and identified by

using TLC and HPLC method . Isolation and

purification Tigogenin compound was made by

using the following steps: Fractionation by

column chromatography, preparative TLC

plates, and purification by using charcoal

material. The identification of isolated

Tigogenin was carried out using melting point ,

Thin layer chromatography , infrared

spectroscopy and HPLC.

References
1. Irish, Gary . Agaves ,Yuccas .and Related

plants : aGardener’s Guide .Timber Press.

2000; .P.1

2. Strecker’s Giant- Skipper Megathymus
streckeri ( Skinner , 1895).Butterflies and

Moths of North America.

3. Dewidar, AM. ; el- Munajjed , D. The
steroid sapogenin constituents of Agave

Americana , A. variegate and Yucca aloifolia

. Planta Med. 1970; 19 (1) : 87-91.

4. Dewik , PM. : Medicinal natural products , a
biosynthetic approach (1

st
ed) . Chapter five ,

The Mevalonate pathway . John Wiley and

Sons Ltd , Chi Chester , reprinted 1997 ; pp .

218-265.

5. Bennett , CC. Public Information Memo .
New York : The Arthritis Founndation , Feb

22, 1977.

6. Cheek , PR. ; Piacent , S. ; Oleszek , W.
Anti- inflmmatory and anti- arthritic effects

of Yucca aloifolia : areview . J Inflamm

(Lond) . 2006 ; 3: 6-13.

7. Woese , C.; Kandler , O. ; Wheelis M.
Towards a natural system of organism :

proposal for the domains archaea , bacteria

and eucarya . Proc Natl Acad Sci USA .

1990; 87 (12) : 4576-4579.

8. Woese, CR .; Fox , GE.Phylogenetic
structure of the prokaryotic domain: the

primary Kingdoms . Proc . Natl. Acad. Sci.

U.S.A. November 1977; 74 (11) : 5088-

5090.

9. Wang , J. ; Chen , j.; yang, KD.and et al .
Study on the production condition of

extraction in combination hydrolysis in situ

for isolation diosgenin . Zhongguo Zhong

Yao Zazhi . 2003; 28 (10) : 934-937.

10. Kemertelidze , EP.; Pkheidze, TA.:
Tigogenin from Yucca glriosa apossible raw

material for the synthesis of steroid

hormonal preparation . Phrmaceutical

Chemistry Journal ,1972;6(12): 795 -797.

11. Wagner, H.; Bladt, S. and Zgainski , EM.:
Plant Drug Analysis, a Thin Layer

Chromatography Atlas . Springer – Verlag ,

Berlin , Germany .1984; p.226.

12. Harborene, JB.; Phytochemical methods ( 1
st Ed.). Chapter two, phenolic compounds .

Chapman and Hall , London , 1976; pp, 115-

117pp.

13. Waksmundzka – Hajnos , M.; Sherma, J.;
Kowalsk,T.: TLC of triterpenes ( including

Saponin). Thin layer Chromatography in

Phytochemistry (1
st
ed) , CRC Press, Taylor

and Francis Group. USA . 2008; PP.528.

14. Shriner, RL.; Hermann, CKF.; Morrill,
TC.: Preliminary examination , Physical

Properties and elemental analysis , The

systematic identificationof organic

compounds (86
th

ed). John Wiley and Sons

Inc., USA. 2004;PP:51-52.