Fitoterapia 74 (2003) 583–591

Furostanol saponins from Tribulus terrestris

E. De Combarieu, N. Fuzzati, M. Lovati, E. Mercalli*
Research and Development Laboratories, Indena S.p.A., Via Don Minzoni 6, Settala (MI) 20090, Italy

Received 22 April 2003; accepted 5 June 2003

Abstract

An HPLC-ELSD-ESI-MS method has been developed for the analysis of the steroidal
saponins in the aerial parts of Tribulus terrestris. Protodioscin, a new saponin (5,6-
dihydroprotodioscin, neoprotodioscin) and their respective sulfates were detected. The
structure of the new compound was elucidated on the basis of NMR and ESI-MS spectral
analysis.
䊚 2003 Elsevier B.V. All rights reserved.

Keywords: Tribulus terrestris; Furostanol saponins; 5,6-Dihydroprotodioscin; Neoprotodioscin; Proto-

dioscin; Prototribestin

1. Introduction

Tribulus terrestris L. (Zygophyllaceae) is an annual herb that grows worldwide,

especially in the subtropical area, used in the folk medicine in India, China, Bulgaria
and other countries against sexual impotency, oedemas, abdominal distention and
cardiovascular diseases. Preparations containing T. terrestris extracts are on sale in
USA as food supplements with claim of a general stimulating action on motor
activity, muscle tone and restorative tonic for vigor. Indeed, T. terrestris preparations
are mainly used to improve performance in sports and for the treatment of impotency
w1–3x. A wide range of compounds has been reported to occur in this plant:
saponins w4–11x; flavonoids w12x; amides and alkaloids w2,13x. Several studies have
shown that saponins are among the compounds responsible for the biological
activities of T. terrestris extracts. Steroidal saponins isolated from T. terrestris are
*Corresponding author. Tel.: q39-02-95413618; fax: q39-02-95413675.
E-mail address: enrico.mercalli@indena.com (E. Mercalli).

0367-326X/03/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0367-326X(03)00152-7
584 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591

spirostanol saponins bearing a sugar chain linked to C-3 and furostanols carrying a
sugar chain at C-3 and a D-glucose residue at C-26. The spirostanols contain mainly
diosgenin, tigogenin and gitogenin as aglycones. Moreover, two new monosulfated
saponins have been recently characterised w8x. In the present paper we report the
development of a qualitative HPLC-ELSD-ESI-MS method to study the distribution
of steroidal saponins in T. terrestris aerial parts. Furthermore, the isolation and
structural characterization of a new dihydro analogue of protodioscin (2) together
with the known saponin protodioscin (1) is described.

2. Materials and methods

2.1. Plant material

T. terrestris aerial parts (origin: Bulgaria) were purchased from Hypokrates

Medicinal Plants (Switzerland) and authenticated by Dr L. Pagni (Botanical
Division, Indena S.p.A.). A voucher specimen (Cy80492) was deposited at the
herbarium of Indena S.p.A., Italy.

2.2. General

HPLC apparatus: a PumpySystem Controller Waters Alliance 2690 with an ELS

detector Sedex 65, S.E.D.E.R.E. Column: X Terra RP 18 (5 mm, 250 mm L=4.6
mm ID) from Waters. Gradient: 0.1% formic acid (vyv) in water (A) and acetonitrile
(B) according to the following profile: 0–10 min, 80–75% A, 20–25% B; 10–20
min, isocratic 75% A, 25% B; 20–40 min, 75–50% A, 25–50% B; 40–41 min,
50–0% A, 50–100% B; 41–45 min, isocratic 0% A, 100% B. Flow rate: 1.0 mly
min. Column temperature, controlled with a column heater–cooler HP series 1100
from Hewlett-Packard, was set at 30 8C. Sedex 65, S.E.D.E.R.E. Evaporative Laser
Scattering Detector conditions were optimized in order to achieve maximum
sensitivity. ELSD conditions: nebulizing gas (N2)s2.5 bars; temperature nebulizing
gass60 8C; gains9.
ESI-MS system: Finnigan MAT LCQ ion trap mass spectrometer equipped with
Microsoft䉸 Windows䉸 NT娃 data system and an electrospray interface (ESI). ESI
positive ion mode conditions: source voltage 5.5 kV, sheath gas flow rate 80 au,
auxiliary gas flow rate 30 au, source current 0.15 mA, capillary voltage 43 V and
capillary temperature 290 8C. ESI negative ion mode conditions: source voltage 3.4
kV, sheath gas flow rate 80 au, auxiliary gas flow rate 30 au, source current 0.15
mA, capillary voltage y4 V and capillary temperature 290 8C. Full scan spectra
from 150 to 2000 u in the negative and positive ion mode were obtained (scan time
1 s). Ion trap conditions: acquisition in automatic gain control with a max-inject
time of 200 ms. The HPLC system included a Finnigan MAT Thermo Separation
Product P4000 pump, a Finnigan MAT SCM 1000 degaser and a Finnigan MAT
AS 3000 auto sampler. The HPLC conditions were as described above.
NMR spectra were recorded in pyridine-d5 on a VARIAN INOVA300 spectrometer
equipped with VNMR娃 Software.
 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591 585

TLC plates: 10=20 cm RP-18 F254S (Merck), detection: UV light and spraying
with Ehrlich reagent. Liquid chromatography: LiChroprep䉸 RP-18 25–40 mm
(Merck).
Solvents and reagents were purchased from Merck.

2.3. Sample preparations and HPLC-MS analysis

Ground plant material (2 g) mixed with 20 ml EtOHyH2 O (2:3) in a glass bottle

with a screw cap, was extracted for 15 min by sonication for 15 min. After
centrifugation (4 h at 200 rev.ymin) the extract was filtered with 0.45 mm PTFE
disposable filter before HPLC injection (injection volume 10 ml).

2.3.1. Compound 1 (protodioscin)

ESI-MS (positive): myzs1031 wMqHyH2 Oxq (C51 H84 O22 requires 1049.24);
885 wMqHyH2OyRhaxq; 869 wMqHyH2OyGlcxq ; 739 wMqHyH2O–
2=Rhaxq; 723 wMqHyH2OyRhayGlcxq ; 577 wMqHyH2Oy2=RhayGlcxq ;
415 wMqHyH2Oy2=Rhay2=Glcxq swgeninqHyH2 Oxq ; ESI-MS (negative):
myzs1047 wMyHxy; 1093 wMyHqHCOOHxy.

2.3.2. Compound 2 (neoprotodioscin)

White amorphous powder; mp 207–208 8C (dec.); IR nmax (KBr) cmy1: 3420,
2933, 1653, 1457, 1382, 1130, 1041, 914, 841, 811; ESI-MS (positive): myzs1033
wMqHyH2Oxq (C51H86O22 requires 1051.24); 887 wMqHyH2OyRhaxq ; 871
wMqHyH2OyGlcxq ; 741 wMqHyH2 Oy2=Rhaxq ; 725 wMqHyH2OyRhay
Glcxq; 579 wMqHyH2Oy2=RhayGlcxq ; 417 wMqHyH2Oy2=Rhay
2=GlcxqswgeninqHyH2Oxq ; ESI-MS (negative): myzs1049 wMyHxy ; 1095
wMyHqHCOOHxy.
1
H-NMR and 13C-NMR spectral data are shown in Tables 1 and 2.

2.3.3. Compound 1a (prototribestin)

ESI-MS (pos.): myzs965 wMqHyH2 Oxq , 885 wMqHyH2OySO3 xq , 819
wMqHyH2OyRhaxq, 803 wMqHyH2OyGlcxq , 739 wMqHyH2OySO3 y
Rhaxq, 723 wMqHyH2OySO3 yGlcxq , 657 wMqHyH2 OyRhayGlcxq , 577
wMqHyH2OySO3yGlcyRhaxq , 415 wMqHyH2OySO3 yRhay2=Glcxq s
wgenqHyH2Oxq; ESI-MS (negative): myzs981 wMyHxy.

2.3.4. Compound 2a (neoprototribestin)

ESI-MS (pos.): myzs967 wMqHyH2 Oxq , 887 wMqHyH2OySO3 xq , 821
wMqHyH2OyRhaxq, 805 wMqHyH2OyGlcxq , 741 wMqHyH2OySO3 y
Rhaxq, 725 wMqHyH2OySO3 yGlcxq , 659 wMqHyH2 OyRhayGlcxq , 579
wMqHyH2OySO3yGlcyRhaxq , 417 wMqHyH2OySO3 yRhay2=Glcxq s
wgenqHyH2Oxq; ESI-MS (negative): myzs983 wMyHxy.
586 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591

Table 1
1
H-NMR spectra data for compound 2

H d (ppm) Multiplicity, J (Hz) GCOSY GHSQC GHMQC

1a 0.96 m 1.65 37.3 37.2
1b 1.74 m 0.85–2.07 (w) 37.3 37.2
2a 1.82 m 2.07 30.0 78.3 (w)
2b 2.07 m 1.74–1.82 30.0 –
3 3.92 m 1.87 77.4 100.0
4a 1.40 m 0.93 39.1 30.2–78.3–44.8
4b 1.87 m 3.92 39.1 30.2–78.3–44.8
5 0.93 br dd 1.15–1.40 44.8 37.2
6a, 6b 1.15 m 0.93 29.0 –
7a 1.48 m 1.56–1.87 32.4 56.5
7b 1.87 m 1.48 32.4 –
8 1.56 br s 1.00–1.48 34.6 50.6
9 0.88 m – 54.6 –
11a, 11b 1.48 m – 21.3 –
12a 1.08 m 1.83 40.3 –
12b 1.83 m 1.08–1.11 (w) 40.3 16.7
14 1.11 m 1.48 56.5 40.8
15a 1.48 m 1.11–2.05–4.94 32.5 81.2
15b 2.05 m 1.48 32.5 41.2–64.9–81.2
16 4.94 m 1.48–1.92 81.2 41.4
17 1.92 s 4.94 64.0 16.4–41.2–81.2
18 0.90 s – 16.7 41.2–56.5–64.0
19 1.04 s – 12.5 37.3–44.8–54.6
20 2.26 m 1.34 40.7 16.4–41.2–64.0–110.6
21 1.34 d (6.9) 2.26 16.4 41.2–64.0–110.6
23a 2.05 m – 37.2 28.4
23b 1.94 m 1.69 37.2 28.4–110.6
24a 2.05 m 1.69 28.4 34.2–37.7
24b 1.69 m 1.94–2.05 28.4 37.7
25 1.94 m 1.01–3.95 34.3 17.5
26a 3.64 dd (5.7–9.4) 3.95 75.2 17.5–28.4–34.3–104.9
26b 3.95 dd (5.7–9.4) 1.94–3.64 75.2 17.5–28.4–34.3–104.9
27 1.01 d (6.5) 1.94 17.5 28.4–34.3–75.2
C-3 sugar part
Glc (inner)
19 4.96 d (7.5) 4.19 100.0 77.9–78.2
29 4.19 m 4.96 77.9 77.9–100.0 (w)–102.1
39 4.20 m 4.33 78.2 77.9–79.2–100.0
49 4.33 m 3.71–4.20 79.2 61.6–76.9–78.2–103.0
59 3.71 m 4.33 76.9 –
69a 4.07 m – 61.6 –
69b 4.15 m – 61.6 –
Rha (1™2)
10 6.31 d (0.9) 4.78 102.1 69.4–72.5–77.9
20 4.78 m 6.31 72.5 72.9–102.1
30 4.56 dd (2.8–9.0) 4.29 72.8 72.5–74.2
40 4.29 m 4.56 74.2 18.5–69.4–72.8–102.1
50 4.92 m 1.74 69.4 18.5–74.2–102.1
60 1.74 d (6.2) 4.92 18.5 69.4–74.2
 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591 587

Table 1 (Continued)
H d (ppm) Multiplicity, J (Hz) GCOSY GHSQC GHMQC
Rha (1™4)
190 5.79 d (1.0) 4.66 103.0 70.5–72.7–79.2
290 4.66 m 4.50–5.79 72.4 72.7–73.9
390 4.50 m 4.25–4.66 72.7 70.5–73.9
490 4.25 m 4.50–4.88 (w) 73.9 18.6–70.5–72.7–102.9
590 4.88 m 1.62–4.25 (w) 70.5 18.6–73.9–103.0
690 1.62 d (6.2) 4.88 18.6 70.5–73.9
C-26 sugar part
Glc
100 4.79 d (7.5) 3.99 104.9 75.3–78.4
200 3.99 m 4.19–4.79 75.3 71.8–78.4–104.9
300 4.19 m 3.99–4.20 78.4 71.8–75.3–78.6
400 4.20 m 4.19 71.8 62.9–75.3–78.4–78.6
500 3.95 m 4.38–4.53 78.6 71.8–104.9
600a 4.38 m 3.95 (w)–4.53 62.9 78.6
600b 4.53 m 3.95 (w)–4.38 62.9 –

2.4. Extraction and analysis

The powdered plant material (2.0 kg) was extracted at room temperature with
40% EtOH. The extract concentrated under vacuum and treated with n-hexane (3=1
l), CHCl3 (3=1 l) and n-BuOH (4=1 l). The BuOH fraction was concentrated to
dryness and the residue (100 g) was dissolved in 200 ml of CH3CNyH2O (15:85
vyv) and CC over 1 kg of LiChroprep䉸 RP-18.
The elution was performed with a solvent gradient starting from CH3CNyH2O
(15:85 vyv) to a final composition of the mobile phase of CH3CNyH2O (35:65
vyv). Eight fractions (F1–F8) were collected. Fraction F4 (10 g) was subjected to
another CC purification on RP-18 packed column wmobile phase CH3CNyH2O
(25:75 vyv)x, obtaining two purified fractions: F4-1 (1, 5 g) and F4-2 (2, 1.2 g).

3. Results and discussion

The presence of steroidal saponins in the ethanol extract of T. terrestris aerial

parts was investigated by means of HPLC-ELSD-ESI-MS. The HPLC-ELSD profile
(Fig. 1) shows the occurrence of four main peaks (1, 2, 1a and 2a). The ESI-MS
spectra of the detected peaks exhibited the wMyHxy and wMyHqHCOOHxy ions
in the negative ionisation mode and the wMqNaxq ion in the positive ionisation
mode. Moreover, the positive ESI-MS spectra displayed an intense wMqHy
H2Oxq ion due to the loss of the labile hydroxyl group in position C-22, typical of
the furostanolic saponins, and signals due to the successive losses of the sugar units
allowing the identification of the compounds. In particular, the presence of signals
at myz 415, 417 was diagnostic for the identification of the dioscin and tigogenin
aglycone, respectively. Therefore, peaks 1 and 1a were identified as the known
compounds protodioscin and prototribestrin w8x, respectively. Peaks 2 and 2a
588 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591

Table 2
13
C-NMR spectra data for compound 2

C d (ppm), multiplicity C d (ppm), multiplicity

1 37.3 (t) C-3 sugar part
2 30.0 (t) Glc (inner)
3 77.4 (d) 19 100.0 (d)
4 39.1 (t) 29 77.9 (d)
5 44.8 (s) 39 78.2 (d)
6 29.0 (d) 49 79.2 (d)
7 32.4 (t) 59 76.9 (d)
8 34.6 (d) 69 61.6 (t)
9 54.6 (d)
10 35.4 (s) Rha (1™2)
11 21.3 (t) 10 102.1 (d)
12 40.3 (t) 20 72.5 (d)
13 41.2 (s) 30 72.8 (d)
14 56.5 (d) 40 74.2 (d)
15 32.5 (t) 50 69.4 (d)
16 81.2 (d) 60 18.5 (q)
17 64.0 (d)
18 16.7 (q) Rha (1™4)
19 12.5 (d) 190 103.0 (d)
20 40.7 (d) 290 72.4 (d)
21 16.4 (q) 390 72.7 (d)
22 110.6 (s) 490 73.9 (d)
23 37.2 (t) 590 70.5 (d)
24 28.4 (t) 690 18.6 (q)
25 34.3 (q)
26 75.2 (t) C-26 sugar part
27 17.5 (d) Glc
100 104.9 (d)
200 75.3 (d)
300 78.4 (d)
400 71.8 (d)
500 78.6 (d)
600 62.9 (t)

displayed ESIq and ESIy spectra with signals possessing 2 m more than those of
peaks 1 and 1a, respectively, suggesting the presence of a furostane-3,22,26-
trihydroxygenin. Therefore, 2 and 2a were tentatively identified as the new 5,6-
dihydro-derivatives of protodioscin and prototribestin, respectively.
In order to confirm the HPLC-MS identification, chromatographic isolation and
NMR investigation of compounds 1 and 2 were carried out. Peak 1 was identified
as protodioscin by comparison of its spectral data with those reported in the literature
w14,15x.
The 1H-NMR spectrum of 2 (Table 1) was very close to that of protodioscin, the
only difference being the presence of a 2H– multiplet centered at d 1.15, and a
one-proton signal at d 0.93 (br dd) instead of the olefinic proton at d 5.30 in 1.
This finding was consistent with a difference of two units in the molecular weight
 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591 589

Fig. 1. HPLC-ELSD chromatogram of the hydroethanolic extracts of T. terrestris.

between 1 and 2, as shown by ESI-MS spectra. The 13C-NMR assignments (Table

2) of the sugar moieties of 2 were achieved by referring to those literature data of
authentic methyl glycosides w16x and were confirmed by GCOSY, GHSQC and
GHMQC spectra. The data also indicated the presence of two terminal rhamnose
units and two glucose units. The b-configuration of the anomeric carbon of glucose
in 2 was proved by the large J1H–2H value ()7.0 Hz). The a-configuration of the
anomeric carbon of the rhamnose was assured by comparison of the chemical shift
values of the carbons 30, 50, 390 and 590 with those of the corresponding carbons of
methyl a- and b-rhamnopyranoside w16x. In GHMQC spectrum, the anomeric proton
signals at d 5.79 (H-190 of the terminal rhamnose), 6.31 (H-10 of the terminal
rhamnose), 4.96 (2,4-substituted glucose attached to C-3) and 4.79 (terminal glucose
attached to C-26) showed cross-peaks with the carbon signals at d 77.9 (C-29 of
the 2,4-substituted glucose attached to C-3), 79.2 (C-49 of the 2,4-substituted glucose
attached to C-3), 77.4 (C-3 of the aglycone) and 75.2 (C-26 of the aglycone).
These signals provide wide evidence to determine the linkages by which the sugars
are connected.
In addition, the furostanolic glycosidic nature of 2 was indicated by the strong
absorption bands at 3420 and 1041 cmy1 and a 25R-furostan steroidal structure
(811, 841 and 914 cmy1, intensity 914-841 cmy1 in the IR spectrum) w17,18x
was confirmed by 1H- and 13C-NMR spectra. Thus, the structure of compound 2
was determined as 26-O-b-D-glucopyranosyl-25(R)-5a-furostan-3b,22a, 26-triol-3-
O- wa- L -rhamnopyranosyl-(1™2)-O-a- L-rhamnopyranosyl-(1™4)x -O-b- D -glu-
copyranside (5,6-dihydroprotodioscin, neoprotodioscin).
The low content of compound 2a in the extract precluded its isolation. However,
the chromatographic behavior and the study of the HPLC-MS spectra recorded on
line allow to reasonably identify 2a as the 5,6-dihydro derivative of prototribestin
(Fig. 2).
590 E. De Combarieu et al. / Fitoterapia 74 (2003) 583–591

Fig. 2. Compounds 1, 1a, 2, 2a.

It is interesting to note that in the investigated Bulgarian T. terrestris no presence

of gitogenin derivatives were detected, differently from what was found in the
analogous plant growing in China, India and South-East Asia.

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