Royleanumioside - A new phytotoxic triterpenoid from Teucrium royleanum

Article abstract of DOI:10.1080/10286020.2015.1006203

Royleanumioside (1), a new lupane type triterpenoid, was secluded from the CHCl₃ soluble fraction of Teucrium royleanum. The structure of royleanumioside (1) was ascertained by spectral studies including 1D and 2D NMR spectroscopy and by the assessment with the reported data in literature. This new identified compound 1 was further tested for its phytotoxic activity against lettuce seedlings. Interestingly royleanumioside (1) had shown moderate phytotoxic activity.

Full text of DOI:10.1080/10286020.2015.1006203

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Royleanumioside – a new phytotoxic
triterpenoid from Teucrium royleanum
Shabir Ahmada, Riaz Ullahb, Naser M. AbdEl-Salamc, Muhammad
Arfand & Hidayat Hussaine
a Department of Chemistry, Islamia College University, Peshawar,
Khyber Pakhtunkhwa 25000, Pakistan
b Department of Chemistry, Government College Ara Khel, FR
Kohat, Khyber Pakhtunkhwa26000, Pakistan
c Riyadh Community College, King Saud University, Riyadh11437,
Saudi Arabia
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d Institute of Chemical Sciences, University of Peshawar,
Peshawar, Khyber Pakhtunkhwa25120, Pakistan
e UoN Chair of Oman's Medicinal Plants and Marine Natural
Products, University of Nizwa, Birkat Al-Mauz, Nizwa616, Oman
Published online: 17 Feb 2015.
To cite this article: Shabir Ahmad, Riaz Ullah, Naser M. AbdEl-Salam, Muhammad Arfan & Hidayat
Hussain (2015): Royleanumioside – a new phytotoxic triterpenoid from Teucrium royleanum, Journal
of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1006203
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1006203
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Journal of Asian Natural Products Research, 2015
http://dx.doi.org/10.1080/10286020.2015.1006203
Royleanumioside – a new phytotoxic triterpenoid from Teucrium
royleanum
Shabir Ahmada, Riaz Ullahb*, Naser M. AbdEl-Salamc, Muhammad Arfand
and Hidayat Hussaine
aDepartment of Chemistry, Islamia College University, Peshawar, Khyber Pakhtunkhwa 25000,
Pakistan; Department of Chemistry, Government College Ara Khel, FR Kohat, Khyber
b
Pakhtunkhwa 26000, Pakistan; cRiyadh Community College, King Saud University, Riyadh 11437,
Saudi Arabia; Institute of Chemical Sciences, University of Peshawar, Peshawar, Khyber
d
e
Pakhtunkhwa 25120, Pakistan; UoN Chair of Oman’s Medicinal Plants and Marine Natural
Products, University of Nizwa, Birkat Al-Mauz, Nizwa 616, Oman
(Received 20 October 2014; final version received 6 January 2015)
Royleanumioside (1), a new lupane type triterpenoid, was secluded from the CHCl3
soluble fraction of Teucrium royleanum. The structure of royleanumioside (1) was
ascertained by spectral studies including 1D and 2D NMR spectroscopy and by the
assessment with the reported data in literature. This new identified compound 1 was
further tested for its phytotoxic activity against lettuce seedlings. Interestingly
royleanumioside (1) had shown moderate phytotoxic activity.
Keywords: Teucrium royleanum; Lamiaceae; lupane type triterpenoid; phytotoxicity
1. Introduction
we have isolated and identified several
compounds. In this study, we account for
the isolation, characterization, and phyto-
toxic activities of one new compound
named royleanumioside (1), a lupane type
triterpenoid.
The genus Teucrium belongs to the family
Lamiaceae and is consisted of 300 species
[1]. Several species of the genus Teucrium
are known for their biological activities
such as antiseptics, antipyretics, and
antispasmodics. Some species of this
genus were also used as tonics [2,3].
Aerial parts of Teucrium spp. showed
hepatotoxic effect when used as decoction
in herbal medicine and this may be due to
the presence of diterpenoids in this genus
[4–7]. Other biological activities reported
from this species are insect antifeedant
activity and antioxidant activity of essen-
tial oil [2,8–11]. Previously some inter-
esting natural products have further been
reported from the aerial parts of Teucrium
royleanum [12]. This background of
T. royleanum incited us for phytochemical
and biological evaluation of this plant.
During the course of our ongoing project,
2. Results and discussion
The chloroform-soluble fraction of the
aerial parts of T. royleanum was subjected
to column chromatography (CC) over
silica gel eluting with different mobile
phases.
Compound 1 was isolated as an
amorphous powder. The HR-FAB-MS
(positive ion mode) provided the molecu-
lar ion peak at m/z 853.6929 [M þ H]þ,
corresponding to the molecular formula
C54H92O7, with nine degrees of unsatura-
tion. The infrared (IR) spectrum showed
strong absorptions at 3360 (OZH), 2900
(CZH), 1740 (CvO), 1630 (CvC), and
*Corresponding author. Email: afridiriaz@yahoo.com
q 2015 Taylor & Francis

2
S. Ahmad et al.
1250 (CZO) cm21. The 1H NMR
spectrum exhibited seven tertiary methyl
singlets at d 0.67, 0.77, 0.85, 0.89, 0.97,
0.99, and 1.54 (all, 3H, s), a trisubstituted
double bond at d 5.33 (1H, t, J ¼ 3.4 Hz).
[14]. The fragments at m/z 208 and 189
indicated a hydroxyl group at ring A or
B. The hydroxyl group was assigned to
C-3 on the basis of HMBC correlations of
H-3 to C-1 and C-2 and was further
confirmed through the characteristic
shifts of C-2 and C-4 in the 13C NMR
spectrum. The b-orientation of the C-3
hydroxyl group was decided by measur-
ing the J values of the double doublet at d
3.57 (1H, dd, J ¼ 9.5, 4.8 Hz) [15]. The
acid hydrolysis of 1 provided various
products among which the glycone was
separated and identified as D-glucose
through its optical rotation sign, retention
time in GC, and by co-TLC with an
authentic sample. The position of the
sugar moiety was confirmed by long
range HMBC experiments in which H-3
1
The H NMR spectrum also showed two
broad doublets for exocyclic double bond
at d 4.70 and 4.62 (1H each, J ¼ 2.0 Hz)
[13]. The signals for sugar moiety
appeared at d 4.35 (1H, d, J ¼ 7.6 Hz, H-
10), 3.32–3.34 (1H, m, H-20), 3.54 (1H, m,
H-30), 3.44–3.46 (1H, m, H-40), 3.41–3.43
(1H, m, H-50), 4.43 (1H, dd, J ¼ 12.0,
4.6 Hz), and 4.25 (1H, dd, J ¼ 12.0,
1.9 Hz). The presence of acyl group was
1
evident from H NMR signals at d 0.65
(3H, t, J ¼ 7.4 Hz), 1.23 (26H, br s), and
2.32 (2H, t, J ¼ 7.4 Hz). The 13C NMR
spectrum (BB and DEPT) of 1 revealed
the presence of 8 methyl, 27 methylene,
11 methine, and 8 quaternary carbon
atoms. It included signals for four olefinic
carbons (d 140.2, 121.4 and 147.4, 111.1),
a hydroxyl-bearing carbon (d 79.4), sugar
moiety (d 101.1, 73.3, 76.2, 70.2, 73.6,
and 63.5), and acyl moiety (d 174.2, 34.1,
24.8, 29.5, and 13.8). A triplet at d 5.33
(1H, J ¼ 3.4 Hz) in the 1H NMR spectrum
and characteristic EI-MS fragments at m/z
216 and 201 resulted from retro-Diels-
Alder cleavage in the ring C indicated the
position of a second double bond at C-12
3
(d 3.57) showed J correlation with the
anomeric carbon (d 101.1) and the
anomeric proton (d 4.35) showed 3J
correlation with C-3 (d 79.4). The
position of aliphatic ester was deduced
at C-60 of the sugar moiety which is due to
the downfield shifts of H-60 (d 4.43 and
4.25), C-6 (d 63.5) and upfield shift of C-
50 (d 73.6) and finally confirmed by
HMBC correlations of H-60 (d 4.43 and
4.25) with C-100 (d 174.2) (Figure 1).
On the basis of these evidences, the
structure of 1 was elucidated as lupa-12,
30
H
29
22
19
20
21
H
18
15
12
26
25
5
11
9
17
16
13
14
28
1
4
2
3
10
8
H
H
27
7
6
6'
O
O
18''
2''
1''
15
O
24
5'
OH
23
O
4'
1
1'
H
HO
2'
3'
OH
Figure 1. Structure and key HMBC correlations of royleanumioside (1).

Journal of Asian Natural Products Research
3
20(29)-dien-60-stearoyl 3-O-b-D-gluco-
pyranoside.
Company, Tokyo, Japan). Ceric sulfate
spray was used for visualization of the TLC
plates under UV at 254 and 366 nm. CC was
carried out using silica gel (70–230 and
230–400 mesh; E-Merck, Darmstadt,
Germany). Aluminum sheets precoated
with silica gel 60 F 254 (0.2 mm thick; E-
Merck) were used for TLC to check the
purity of the compounds and were visual-
ized under UV light (254 and 366 nm)
followed by ceric sulfate as the spray
reagent.
The results of the phytotoxic bioassay
against lettuce seedlings (Lactuca sativa
cv. Great Lakes 366) are reported in
Figure 2. Compound 1 exhibits moderate
phytotoxicity. The effects produced by our
tested compound 1 on the roots inhibition
are presented in Figure 2. The overall effect
on L. sativa inhibition was 22–28.6%,
while interestingly low inhibition had been
shown at higher concentration and none of
the concentration has an EC50 value. The
available data on the lupanic triterpenes
showed stimulation of the germination
of L. sativa [16]. The saponins are
another class of closely related secondary
metabolites exhibiting slight inhibitory
activities [17].
3.2 Plant material
The aerial parts of the plant T. royleanum
were collected in June 2003 from Swat
(Pakistan) and deposited in the Depart-
ment of Botany with voucher specimen
[No. Shabir 2651979(PUP)]. It was ident-
ified by Plant Taxonomist Professor Dr
A. Rashid, Botany Department, University
of Peshawar, Pakistan.
3. Experimental
3.1 General experimental procedures
JASCO DIP-360 Polarimeter(JASCO, Inc.,
Easton, MD, USA) was used to determine
optical rotations. IR spectrum was recorded
on 460 Shimadzu Spectrometer (Shimadzu,
Tokyo, Japan). The 1H, 13C NMR, and
HMBCspectraweretakenonBrukerNMR-
spectrophotometer (Bruker Instruments,
Inc., Karlsruhe, Germany) operating at
3.3 Extraction and isolation
The dried and powdered whole plant
(10 kg) of T. royleanum was exhaustively
extracted in MeOH for 3 weeks and
filtration and evaporation of the solvent
afforded a crude extract (550 g), which
was extracted with n-hexane, CHCl3, and
n-BuOH. The chloroform extract (50 g)
was subjected to CC on silica gel using
1
400 MHz for H NMR and 100 MHz for
13C NMR. Mass spectra were recorded on
JMS-DA 500 mass spectrometers (JEOL
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
R
H
0.0
3
10
30
100
300
Concentration (ppm)
Figure 2. Inhibitory effects of royleanumioside (1) (R, root; H, hypocotyl).

4
S. Ahmad et al.
n-hexane, n-hexane–CHCl3, CHCl3 –
MeOH and finally, pure MeOH as mobile
phases which yielded 11 fractions. Frac-
tion no. 7 of the first column was loaded on
silica gel and eluted with MeOH–CHCl3
(1:9) to give compound 1 (11 mg).
bath. The reaction mixture was evapor-
ated, and the hydrolysate after dilution
with H2O (10 ml) was extracted with
CHCl3 (10 ml £ 3). The CHCl3 extracts
were evaporated to afford glycone which
was identified as D-glucose by the sign of
25
its optical rotation (½aꢀD þ 51.8
(c ¼ 0.02, MeOH)), retention time in GC
(Rt ¼ 11.481), and by co-TLC with an
authentic sample.
3.3.1 Royleanumioside (1)
25
Colorless amorphous solid; ½aꢀD 234
(c ¼ 1.2, CHCl3); IR nmax cm21 (KBr):
1
3360, 2900, 1740, 1630, 1250. H NMR
3.5 Phytotoxicity bioassay
(CD3OD): 5.33 (1H, t, J ¼ 3.4 Hz, H-12),
4.70 (1H, d, J ¼ 2.0 Hz, H-29a), 4.62 (1H,
d, J ¼ 2.0 Hz, H-29b), 4.43 (1H, dd,
J ¼ 12.0, 4.6 Hz, H-60), 4.35 (1H, d,
J ¼ 7.6 Hz, H-10), 4.25 (1H, dd, J ¼ 12.0,
1.9 Hz, H-60), 3.57 (1H, dd, J ¼ 9.5, 4.8 Hz,
H-3), 3.53–3.55 (1H, m, H-30), 3.44–3.46
(1H, m, H-40), 3.41–3.43 (1H, m, H-50),
3.32–3.34 (1H, m, H-20), 2.75–2.77 (1H,
m, H-19), 2.32 (2H, t, J ¼ 7.4 Hz, H-200),
1.59–1.61 (2H, m, H-300), 1.54 (3H, s, CH3-
30), 1.23 (26H, br s, H-400-H-1600), 0.99 (3H,
s, CH3-27), 0.97 (3H, s, CH3-24), 0.89 (3H,
s, CH3-23), 0.85 (3H, s, CH3-28), 0.77 (3H,
s, CH3-25), 0.67 (3H, s, CH3-26), 0.65 (3H,
t, J ¼ 7.4 Hz, H-1800). 13C NMR (CD3OD):
174.2 (C-100), 147.4 (C-22), 140.2 (C-13),
121.4 (C-12), 111.1 (C-29), 101.1 (C-10),
79.4 (C-3), 76.2 (C-30), 73.6 (C-50), 73.3 (C-
20), 70.2 (C-40), 63.5 (C-60), 56.6 (C-18),
55.9 (C-5), 50.0 (C-9), 49.3 (C-19), 42.1
(C-4), 42.0 (C-14), 39.6 (C-21), 38.6 (C-1),
36.5 (C-10), 36.4 (C-17), 34.1 (C-200), 31.8
(C-11), 31.7 (C-7), 29.5 (C-400 –C-1600),
29.0 (C-16), 27.9 (C-15), 24.8 (C-300), 24.1
(C-2), 22.5 (C-6), 19.1 (C-27), 18.5 (C-30),
18.4 (C-24), 17.5 (C-23), 13.9 (C-28), 13.8
(C-1800), 11.8 (C-25), 11.6 (C-26). El-MS:
m/z 423 (15), 407 (10), 216 (20), 208 (25),
201 (17), 189 (34), 69 (24), 175 (19). HR-
A filter paper (27 mm Ø, type 1, Toyo
Roshi Kaisha, Ltd, Tokyo, Japan) was
placed in a glass petri dish (27 mm Ø),
followed by the addition of a test solution
to the filter paper in the petri dish and
drying completely in vacuo at 408C. After
addition of distilled water (0.7 ml), six pre-
germinated (16 h at 208C in the dark)
seedlings of lettuce (L. sativa cv. Great
Lakes 366) were placed on the filter paper,
and incubated for 48 h at 208C in the dark.
The inhibitory activity on root elongation
was detected by measuring the length of
the root and comparing the data obtained
with that of untreated controls.
Disclosure statement
No potential conflict of interest was reported
by the authors.
Acknowledgement
The authors are thankful to the Deanship of
Scientific Research, King Saud University,
Riyadh, Saudi Arabia, for funding the work
through the research Group project No. RGP-
210.
References
þ
FAB-MS: m/z 853.6929 [M þ H] (calcd
[1] S.I. Ali and Y.J. Nasir, Flora of Pakistan
(Royal Botanic Garden, Edinburgh,
1990), Vol. 192, pp. 17–25.
for C54H93O7, 853.6921).
[2] C.
Cavaleiro,
L.R.
Salgueiro,
M.G. Miguel, and A.P. Proencꢀa da
cunha, J. Chromatogr. A 1033, 187
(2004). doi: 10.1016/j.chroma.2004.01.
005.
3.4 Acid hydrolysis of 1
Compound 1 was refluxed with 2 M HCl in
MeOH (5 ml) at 808C for 2 h in a water

Journal of Asian Natural Products Research
5
[3] M.M.A. Hassan, F.J. Muhtadi, and A. Al-
Badr, J. Pharm. Sci. 68, 800 (1979). doi:
10.1002/jps.2600680639.
[4] E. Mazokopakis, S. Lazaridou, M. Tzardi,
J. Mixaki, I. Diamantis, and E. Ganotakis,
Phytomedicine 11, 83 (2004). doi: 10.
1078/0944-7113-00337.
Soundarya devi, S.S. Rajan, R. Malathi,
and K. Ravikumar, Phytochemistry 64,
1119 (2003). doi: 10.1016/S0031-9422(03)
00510-7.
[11] V. Saroglou, M. Arfan, A. Shabir, D.
Hadjipavlou-litina, and H. Skaltsa, Fla-
vour Frag. J. 22, 154 (2007). doi: 10.
1002/ffj.1774.
[12] M. Hasan, M.D. Ramzan, C. Maria, and
G. Alvarez, Fitoterapia 6, 55 (1993).
[5] D. Polymeros, D. Kamberoglou, and
V. Tzias, J. Clin. Gastroenterol. 34, 100
(2002).
200201000-00022.
doi:
10.1097/00004836-
[13] V.U.
Ahmad,
S.
Bano,
and
[6] J. Perez Alvarez, F. Saez-Royuela, E.
Gento Pena, A. Lopez Morante, A.
Velasco Oses, J. Martin Lorente, Gastro-
entrol. Hepatol. 24, 240 (2001). doi: 10.
1016/S0210-5705(01)70162-8.
F.V. Mohammad, Planta Med. 51, 521
(1985). doi: 10.1055/s-2007-969582.
[14] H. Budzikiewicz, J.M. Wilson, and C.
Djerassi, J. Am. Chem. Soc. 85, 3688
(1963). doi: 10.1021/ja00905a036.
[7] D. Larrey, T. Vial, and A. Pauwels, Ann.
Intern. Med. 117, 129 (1992). doi: 10.
7326/0003-4819-117-2-129.
[8] S. Nakanishi, E. Koreeda, S. Goto,
T. Matsuoka, M. Koreeda, and K.
Nakanishi, J. Am. Chem. Soc. 92, 7512
(1970). doi: 10.1021/ja00728a068.
[15] H. Kizu, M. Koshijima, M. Hayashi,
and T. Tomimori, Chem. Pharm. Bull.
33, 1400 (1985). doi: 10.1248/cpb.33.
1400.
´
[16] F.A. Macıas, A.M. Simonet, and
M.D. Esteban, Phytochemistry 36, 1369
(1994). doi: 10.1016/S0031-9422(00)
89726-5.
´
[9] J. Coll and Y. Tandron, Phytochemistry
65, 387 (2004). doi: 10.1016/j.phytochem.
2003.09.018.
[10] G.N.K. Krishna kumari, S. Aravind, J.
Balachandran, M.R. Ganesh, D.S.
[17] G.R. Waller, M. Jurzysta, and
R.L.Z. Thornf, Hot. Bull. Acad. Sin.
34, 1 (1993).