Separation and identification of anew saponin from the flowers of Guaiacum officinale L.

Article abstract of DOI:10.1080/14786410902884693

A triterpenoid saponin, guaianin O (1), oleanolic acid 3-O-{α-L- rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 3)]-α-L-arabinopyranoside}-28-O-[β-D-glucopyranosyl]-ester, was isolated from the n-butanol extract of flowers of Guaiacum officinale L. The structural elucidation of 1 was accomplished by extensive studies of both one and two dimensional ¹H, ¹³C-NMR spectra, the FAB mass spectrum, and alkaline and acid hydrolyses.

Full text of DOI:10.1080/14786410902884693

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Separation and identification of a new
saponin from the flowers of Guaiacum
officinale L.
Nikhat Saba a , Viqar Uddin Ahmad b , Zulfiqar Ali b & Khalid
Mohammed Khan b
a Department of Chemistry, Jinnah University for Women, V-c,
Nazimabad Karachi, Pakistan
b HEJ Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi,
Karachi-75270, Pakistan
Version of record first published: 24 Nov 2010
To cite this article: Nikhat Saba, Viqar Uddin Ahmad, Zulfiqar Ali & Khalid Mohammed Khan (2010):
Separation and identification of a new saponin from the flowers of Guaiacum officinale L., Natural
Product Research: Formerly Natural Product Letters, 24:20, 1877-1882
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Natural Product Research
Vol. 24, No. 20, 15 December 2010, 1877–1882
Separation and identification of a new saponin from the
flowers of Guaiacum officinale L.
b
Nikhat Sabaa, Viqar Uddin Ahmadb , Zulfiqar Ali
*
and Khalid Mohammed Khanb
aDepartment of Chemistry, Jinnah University for Women, V-c, Nazimabad Karachi,
Pakistan; HEJ Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan
b
(Received 11 March 2009; final version received 13 May 2009)
A triterpenoid saponin, guaianin O (1), oleanolic acid 3-O-{ꢀ-L-rhamno-
pyranosyl-(1 ! 2)-[ꢁ-D-glucopyranosyl-(1 ! 3)]-ꢀ-L-arabinopyranoside}-
28- O-[ꢁ-D-glucopyranosyl]-ester, was isolated from the n-butanol extract
of flowers of Guaiacum officinale L. The structural elucidation of 1 was
accomplished by extensive studies of both one and two dimensional 1H,
13C-NMR spectra, the FAB mass spectrum, and alkaline and acid
hydrolyses.
Keywords: guaianin O; triterpenoid bidesmosidic saponin; flowers;
Guaiacum officinale L.; Zygophyllaceae
1. Introduction
Guaiacum officinale L. is both a medicinal and an ornamental plant and belongs
to the family Zygophyllaceae (Ghafoor, Nasir, & Ali, 1974). The resin of G. officinale
is used as medicine for a number of diseases, e.g. in the beginning stages of angina,
tonsillitis, rheumatoid arthritis, mucous membrane diseases and in abnormalities of
metabolic processes. It is diuretic, diaphoretic, sudorific and sialogogic. It is also
a good antioxidant and antiinflammatory agent (Duwiejua, Zeitlin, Waterman, &
Gray, 1994; Hoppe, 1975). A chemical literature survey revealed the presence of both
mono and bidesmosidic saponins having akebonic and oleanolic acids as genins
(Ahmed, Bano, Fatima, & Bano, 1988; Tori, Seo, Shimaoka, & Tomita, 1974).
The compound 1 was isolated as glass.
2. Results and discussion
Guaianin O was isolated from the n-BuOH extract of the flowers of G. officinale.
The n-BuOH extract was subjected to repeated column and flash chromatography.
The fractions, eluted with 20% methanol in chloroform, were finally purified
by HPLC using a reverse phase C18 column. The compound (1) was purified from
*Corresponding author. Emails: vuahmad@iccs.edu; vuahmad@hotmail.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410902884693
http://www.informaworld.com

1878
N. Saba et al.
among a number of reported compounds (Ahmed et al., 1988; Ahmed, Perveen, &
Bano, 1990).
The FABMS of 1 indicated the loss of masses of 132, 146 and 162 due to the loss
of arabinose, rhamnose and glucose from molecule, respectively (Figure 1). These
sugars were identified by the co-TLC comparison of the water layer of the acid
hydrolysate of 1 with the authentic sugar samples. The pseudomolecular ion peak at
m/z 1057 [MꢀH]ꢀ, molecular weight 1058, corresponding to the molecular formula
C53H86O21 showed the presence of 11 degrees of unsaturation in the molecule.
The next ion peak at m/z 895, [(MꢀH)-162]ꢀ showed the removal of an ester glucose.
The other ion peaks at m/z 749, [(MꢀH)-162-146]ꢀ, 733 (small) [(MꢀH)-162-162]ꢀ,
indicated branching in the sugar chain, 587 [(MꢀH)-162-146-162]ꢀ and 455 [(MꢀH)-
162-146-162-132]ꢀ or [oleanolic acid–H]ꢀ indicated the removal of terminal
rhamnose and glucose as well as internal arabinose, respectively. The fragment
m/z 455 indicated the presence of aglycon, oleanolic acid, molecular weight 456 and
molecular formula C30H48O3. The alkaline hydrolysis followed by FABMS of the
butanol layer showed m/z 895, the absence of an ester bond sugar glucose.
In 1H-NMR (500 MHz, C5D5N) spectra, the protons of each of the seven methyl
groups of the aglycon appeared as singlets at ꢂ 1.17, 1.09, 0.83, 1.06, 1.23, 0.88 and
0.86 of 3H for each of H-23, H-24, H-25, H-26, H-27, H-29 and H-30, respectively.
The H-12 proton appeared as a triplet at ꢂ 5.39 (J ¼ 3.45 Hz) and the H-3 as an
1
overlapped signal at ꢂ 3.57. In the H-NMR spectrum, the four-anomeric proton
29
30
21
22
20
19
12
26
13
11
18
25
10
17
16
C
28
O
1
4
14
9
2
O
8
15
3
27
7
5
6
455
23
24
OH
O
O
5'
1'
4'
O
2'
3'
OH
6''''
O
5''''
587
O
1''''
749
OH
4''''
H
OH
3''''
5''
2''''
OH
O
HO
1''
CH
733
6'''
3
4''
OH
2''
3''
HO
OH
O
5'''
4'''
OH
1'''
2'''
OH
3'''
OH
895
Figure 1. Positions and FAB negative m/z 1057 of 1.

Natural Product Research
1879
signals appeared at ꢂ 4.84 (H-10), 6.11 (H-100), 5.07 (H-1000) and 6.30 (H-10000). The first
and last two signals appeared as doublets, while H-100 appeared as broad singlets due
10–20
to the overlapping of the two signals. The J-values were calculated as J
5.50 Hz, J1
¼
000ꢀ2000
0000ꢀ20000
¼ 6.8 Hz and J1
¼ 8.0 Hz. These coupling constants indicated
the ꢁ-D-pyranosyl configuration for glucose and ꢀ-L-pyranosyl for arabinose and
rhamnose, respectively. The methyl protons of rhamnose resonated as a doublet at
ꢂ 1.60 (J ¼ 6.15 Hz). The study and the comparison of all the above accumulated data
proved that the aglycon of 1 was oleanolic acid. The 13C-NMR (125 MHz, C5D5N)
(BB) of 1 showed that 30 carbons out of 53 consisted of aglycon in the molecule.
The DEPT pulse sequence showed the presence of 7 methyl, 10 methylene and 5
methyne groups. The eight quaternary carbon atoms were detected by the broadband
13C-NMR spectrum. The peaks identified an endocyclic double bond between the
C-12 and C-13 appeared at ꢂ 122.8 (CH) and 144.0 (quaternary carbon atom),
respectively (Ahmed et al., 1988; Tori et al., 1974). A slight low frequency shift of
C-28, ꢂ 176.40 and its IR absorption at 1740 cmꢀ1 suggested a glycosidic linkage
occurred at it. The attachment of polysaccharide chain at C-3 of aglycon was
confirmed by the 10 ppm high frequency chemical shift ꢂ 88.07 (Ahmed et al., 1988;
Tori et al., 1974). Hence it was a 3,28-bidesmosidic compound (Ahmed et al., 1988,
1990). The signals for the seven-methyl groups of the aglycon appeared at ꢂ 27.9,
16.95, 15.58, 17.39, 25.88, 32.93 and 23.57 for the C-23-C-27, C-29 and C-30,
respectively, in the 13C-NMR spectrum. The remaining 23 carbon signals out of 53
were assigned to the four sugar moieties attached to the C-3 and C-28 carbons of
aglycon. The three-anomeric carbon signals of the sugars at the C-3 carbons of
aglycon appeared at ꢂ 104.48 (C-10), 101.72 (C-100) and 104.39 (C-1000) for arabinose,
rhamnose and glucose, respectively. A 10 ppm low frequency chemical shift ꢂ 95.86
(C-10000) was observed for the anomeric carbon of the glucose moiety attached at the
C-28 carbonyl carbon of aglycon as ester linkage. We found that four carbon signals
around ꢂ 78-79 appeared for the two C-3000, C-30000 and two C-5000, C-500000 of the two
terminal glucose moieties. The high frequency chemical shifts of C-2 and C-30 of
arabinose ꢂ 74.74 and 81.78 were due to the attachment of C-100 and C-1000 of terminal
rhamnose and glucose to those positions. The 13C-NMR values of the three terminal
sugars, two glucose and one rhamnose, were assigned with the help of one- and two-
dimensional-NMR spectra and compared to reported data (Ahmad, Saba, Ali,
Muhammad, & Alam, 2000; Ahmad & Saba, 1993; Ahmad, Saba, & Khan, 2004;
Ahmed et al., 1988; Seo, Tomita, Tori, & Yoshimura, 1978). The points of
attachment of the two terminal sugars, rhamnose and glucose, to the internal
arabinose were confirmed via COSY-45ꢁ and HMBC. The sugar linkages and their
13C-NMR data of 1 were in agreement with the reported compound except the
aglycon part (Ahmed et al., 1990).
The interglycosidic linkages were confirmed by the study of COSY-45ꢁ and were
verified by HMBC (Figure 2). The HMBC spectrum showed that the H-10 signal at
ꢂ 4.84 showed a long range connectivity with the carbons C-3, C-20, C-30 and C-50
confirmed that arabinose was directly attached to the C-3 of aglycon and its C-20
and C-30 had high frequency 13C-NMR chemical shift. The H-100 proton ꢂ 6.11 showed
long-range connectivity with the carbons C-20, C-200, C-300 and C-500, verifying the
(C-100-C-20) glycosidic linkage between rhamnose and arabinose. The anomeric proton
H-1000 ꢂ 5.07 showed connectivity with C-30, C-2000, C-3000 and C-5000, which confirmed
a (C-1000-C-30) glycosidic linkage between glucose and arabinose. The anomeric proton

1880
N. Saba et al.
H
C
O
O
H
OH
O
OH
O
O
O
OH
O
O
OH
OH
H
H
OH
OH
OH
H
OH
OH
O
CH
3
H
OH OH
Figure 2. HMBC interactions of 1.
H-10000 (ꢂ 6.30) showed coupling activity with the carbons C-28, C-20000, C-30000 and C-50000.
These linkages were also compared by the reported data (Ahmed et al., 1990). Based
on all of the above accumulated data, the structure of compound 1 has been
determined as oleanolic acid 3-O-{ꢀ-L-rhamnopyranosyl-(1 ! 2)-[ꢁ-D-glucopyrano-
syl-(1 3)]-ꢀ-L-arabinopyranoside}-28- O-[ꢁ-D-glucopyranosyl]-ester.
3. Experimental
3.1. General details
Melting points were determined in glass capillary tubes using a Gallenkamp melting
point apparatus. Optical rotation was measured using a Jasco DIP-360 automatic
digital polarimeter. The IR spectrum was recorded on a Jasco A-100 spectro-
photometer. The UV-spectrum was recorded in methanol on Shimadzu UV 254.
The one- and two-dimensional 1H and 13C-NMR spectra were recorded on a Bruker
AM-500 spectrometer at 500 and 125 MHz, respectively, on the same instrument.
Mass spectra were recorded as fast atom bombardment (FAB) measurements, which
were performed on a MAT 312 mass spectrometer. HPLC was performed on a
Shimadzu apparatus.
3.2. Plant material
The flowers of G. officinale L., 5 kg (fresh), were collected from the premises
of Karachi University in April and were identified by Dr M. Qaiser; a voucher
specimen is deposited in the Herbarium of the Department of Karachi University,
Pakistan (no. 33 KUH).

Natural Product Research
1881
3.3. Extraction and purification of 1
The air-dried and ground material of the flowers of G. officinale (5 kg fresh)
was extracted with distilled with methanol (5 L ꢂ 3) at room temperature and then
concentrated. The resulting brown, thick residue was dissolved in water, extracted
with ethyl acetate followed by extraction with n-butanol (1.5 L). The combined
n-butanol layer was concentrated (9 g) and subjected to repeated column
chromatography and flash chromatography on silica gel with chloroform,
chloroform : methanol and methanol. The fraction eluted with methanol : chloroform
(20 : 80, v/v) contained a mixture of compounds. They were separated and purified by
HPLC having the solvent system methanol : water (30 : 70, v/v) using an RP C18 semi
preparative bondapak column. Compound 1 was purified via HPLC using 29%
water in methanol and obtained as glass, m.p. 360ꢁ–365ꢁC (decomposed). [ꢀ]D25
ꢁ
+ 1
3
(c 0.85, CH OH).
1H-NMR (500 MHz, C5D5N): ꢂ 0.83, s, 3H, (CH3); 0.86, s, 3H, (CH3); 0.88, s,
3H, (CH3); 1.06, s, 3H, (CH3); 1.09, s, 3H, (CH3); 1.17, s, 3H, (CH3); 1.23, s, 3H,
(CH3); 3.57, overlapped, 1H, C3-H; 5.39, t, J 3.45 Hz, 1H, C12-H; 4.84, d, J ¼ 5.5 Hz,
1H, C10-H; 4.62, m, 1H, C20-H; 4.30, m, 1H, C30-H; 4.51, m, 1H, C40-H; 4.22, m, 2H,
C50-H; 6.11, b.s., 1H, C100-H; 4.69, m, 1H, C200-H; 4.56, m, 1H, C300-H; 4.25, m, 1H,
C400-H; 4.55, m, 1H, C500-H; 1.60, d, J ¼ 6.15, 3H, C600-H; 5.07, d, J ¼ 6.8 Hz, 1H,
C1000-H; 3.92, m, 1H, C2000-H; 4.16, m, 1H, C3000-H; 4.33, m, 1H, C4000-H; 4.01, m, 1H,
C5000-H; 4.43, m, 2H, C6000-H; 6.30, d, J ¼ 8.0 Hz, 1H, C10000-H; 4.18, m, 1H, C20000-H;
3.88, m, 1H, C30000-H; 4.13, m, 1H, C40000-H; 4.27, m, 1H, C50000-H; 4.45, m, 2H,
C60000-H.
13C-NMR (125 MHz, C5D5N): ꢂ (C 1) 38.82; (C-2) 26.40; (C-3) 88.07; (C-4) 46.92;
(C-5) 55.79; (C-6) 18.43; (C-7) 33.30; (C-8) 39.8; (C-9) 47.86; (C-10) 39.49; (C-11)
23.32; (C-12) 122.80; (C-13) 144.00; (C-14) 42.05; (C-15) 28.16; (C-16) 23.71; (C-17)
49.97; (C-18) 41.53; (C-19) 45.57; (C-20) 30.68; (C-21) 38.82; (C-22) 33.80; (C-23)
16.95; (C-24) 27.90; (C-25) 15.58; (C-26) 17.39; (C-27) 25.88; (C-28) 176.40; (C-29)
32.93; (C-30) 23.57; (C-10) 104.48; (C-20) 74.74; (C-30) 81.78; (C-40) 67.91; (C-50)
64.55; (C-100) 101.72; (C-200) 72.28; (C-300) 72.44; (C-40) 73.82; (C-500) 69.96; (C-600)
18.38; (C-1000) 104.39; (C-2000) 74.86; (C-3000) 78.11; (C-4000) 70.91; (C-5000) 79.21; (C-6000)
62.13; (C-10000) 95.86; (C-20000) 74.04; (C-30000) 78.46; (C-40000) 71.34; (C-50000) 78.80;
(C-600) 62.45.
FAB mass spectrum (negative mode): m/z, 1057 [MꢀH]ꢀ, 895 [MꢀH-162]ꢀ, 749
[MꢀH-162-146]ꢀ, 733 (small) [MꢀH-162-162]ꢀ 587 [MꢀH-162-146-162]ꢀ, 455
[MꢀH-162-146-162-132]ꢀor [aglyconꢀH]ꢀ.
UV (MeOH) jmax: 202 nm; IR ꢃmax (KBr): 3450 (OH), 2910 (C–H) 1740 (C¼O), 1620
(C¼C) and 1100–1000 cmꢀ1 (C–O–C).
3.4. Acid hydrolysis of 1
About 10 mg of compound 1 was refluxed with dioxane : water : hydrochloric acid
(1 : 1 : 1.5) for 3 h. The reaction mixture was concentrated under reduced pressure to
remove methanol. Water was added. The residue of aglycon was filtered and washed
with water. The aglycon, oleanolic acid, was crystallised and compared with an

1882
N. Saba et al.
authentic sample, m.p. 302–304ꢁC. It was treated with diazomethane to obtain a
methyl ester, m.p. 196–198ꢁC. The aqueous layer was evaporated under reduced
pressure. The sugars were identified as arabinose, rhamnose and glucose by
comparison with authentic samples on cellulose TLC. The TLC solvent system
consisted of ethyl acetate : water : methanol : acetic acid (65 : 15 : 15 : 20). Spots were
detected by spraying it with aniline phthalate.
3.5. Alkaline hydrolysis of 1
About 10 mg of 1 was dissolved in 5 mL methanol and 5 mL solution of 20% sodium
hydroxide was added. The whole mixture was refluxed for 2 h on water bath and then
neutralised with Dowex 50. It was extracted with n-butanol. The aqueous layer was
concentrated under reduced pressure and the residue was identified as glucose by
comparing it with an authentic sample on cellulose TLC plate, developed in ethyl
acetate : water : methanol : acetic acid (65 : 15 : 15 : 20). Spots were detected by
spraying the TLC plate with aniline phthalate. The n-butanol extract was also
concentrated under reduced pressure. The FAB negative ion mass spectrum of the
prosapogenin of 1 indicated ion peaks at m/z 895, 749, 733, 587 and 455, indicating
the loss of only one glucose moiety.
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