A new oxo-sterol derivative from the rhizomes of Costus speciosus

Article abstract of DOI:10.1080/14786419.2017.1324962

Chemical investigation of the rhizomes of Costus speciosus led to the isolation of a new compound, 22-ketocholesteryl palmitate (1) along with four known compounds, 24-methylenecycloartanol (2), cycloartanol (3), stigmasterol (4) and linoleic acid (5). The structure of new compound was characterised by extensive 1D-, 2D-NMR and mass spectrometry (GC-MS and HR-ESI-MS) techniques.

Full text of DOI:10.1080/14786419.2017.1324962

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new oxo-sterol derivative from the rhizomes of
Costus speciosus
Ashish Kumar, Gopi Chand & Vijai K. Agnihotri
To cite this article: Ashish Kumar, Gopi Chand & Vijai K. Agnihotri (2017): A new oxo-
sterol derivative from the rhizomes of Costus speciosus, Natural Product Research, DOI:
10.1080/14786419.2017.1324962
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Date: 08 May 2017, At: 05:08

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1324962
A new oxo-sterol derivative from the rhizomes of Costus
speciosus
Ashish Kumara,b, Gopi Chandc and Vijai K. Agnihotria,b
aacademy of scientific and Innovative research, csIr-Institute of himalayan Bioresource technology,
Palampur, India; bNatural Product chemistry and Process development division, csIr-Institute of himalayan
Bioresource technology, Palampur, India; cBiodiversity division, csIr-Institute of himalayan Bioresource
technology (council of scientific and Industrial research), Palampur, India
ABSTRACT
ARTICLE HISTORY
received 6 February 2017
accepted 22 april 2017
Chemical investigation of the rhizomes of Costus speciosus led to
the isolation of a new compound, 22-ketocholesteryl palmitate (1)
along with four known compounds, 24-methylenecycloartanol (2),
cycloartanol (3), stigmasterol (4) and linoleic acid (5). The structure
of new compound was characterised by extensive 1D-, 2D-NMR and
mass spectrometry (GC-MS and HR-ESI-MS) techniques.
KEYWORDS
Zingiberaceae; Costus
speciosus; 22-Ketocholesteryl
palmitate
1. Introduction
Costus speciosus Koen. (Family: Zingiberaceae), an esteemed Indian ornamental plant, has
been extensively used in oriental systems of medicine. The rhizomes of this plant are
attributed to be bitter, astringent, acrid, cooling, aphrodisiac, purgative, anthelmintic,
depurative, febrifuge, expectorant, tonic, improves digestion and as stimulant herb that
clears toxins (Deni 2008; Gupta 2010). C. speciosus is well known to exhibit diverse
CONTACT Vijai K. agnihotri
IhBt Publication Number: 4028
kantvijai@yahoo.com, vijai@ihbt.res.in
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1324962.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
A. KUMAR ET AL.
O
21
18
24
22
20
17
27
25
26
23
12
11
19
13
14
16
1
4
9
2
O
1'
15
10
8
7
13'
5'
15'
11'
9'
7'
3;
3
5
O
6
14'
12'
10'
8'
4'
6'
2,
16'
1
HO
HO
HO
4
3
2
O
OH
5
Figure 1. structure of isolated compounds from C. speciosus rhizomes.
biological activities, including antidiabetic, hypolipidemic, anticholinesterase, hepatopro-
tective, antibacterial and antifungal (Srivastava et al. 2011). Isolation of oxo acids, branched
fatty acid esters, sesquiterpene lactones, steroids, steroid glycosides, saponins such as
diosgenin, dioscin and gracillin as major constituents was reported from the rhizomes of
C. speciosus (Srivastava et al. 2011; Al-Attas et al. 2015). Diosgenin isolated from rhizomes
of C. speciosus also displayed excellent anti-inflammatory, antioxidant and anti-angiogenic
activities (Selim & Al Jaouni 2016). In continuation to our previous studies on isolation of
compounds from western Himalayan regions (Agnihotri et al. 2014; Thakur & Agnihotri
2016; Walia et al. 2016), in present work, we had isolated one new compound, 22-keto-
cholesteryl palmitate (1) along with four known compounds, 24-methylenecycloartanol
(2), cycloartanol (3), stigmasterol (4) and linoleic acid (5) from rhizomes of Costus speciosus
(Figure 1).
2. Results and discussion
Compound 1 was obtained as white solid. Its molecular formula was determined as C43H74O3
on the basis of HR-ESI-MS which had also suggested seven degrees of unsaturation. The 13
C
spectrum of this compound showed the presence of signals for one double bond, one carbonyl
and one carboxyl group. Presence of peaks at 14.0, 22.6, 25.0, 31.9, 34.6, 173.2 along with group
of peaks in between 29.0 and 29.6 are characteristic for the presence of a long fatty acid chain.
Hence this molecule should be an ester of long fatty acid chain. Long fatty acid chain was
characterised as palmitic acid by preparation of its methyl ester (Agnihotri et al. 2009) followed
by comparison of fragmentation pattern in GC-MS (Adams 2007). After deletion of fatty acid

NATURAL PRODUCT RESEARCH
3
characteristics from 13C spectra, the remaining peaks suggested the molecule should have
another tetracyclic structure with five methyls, ten methylenes, eight methines and four qua-
ternary carbons.The comparison of NMR data of compound (1) with previous literature strongly
indicated another moiety of this molecule as cholesterol (Table S1) (Plouguerné et al. 2006;
Augustin et al. 2015). The methyl signal at δH 0.70 (3H, s, Me-18) showed its HMBC correlations
with C-12 (δC 39.5), C-13 (δC 42.4), C-14 (δC 55.9) and C-17 (δC 52.0), whereas, methyl at δH1.02
(3H, s, Me-19) confirmed by its HMBC correlation with C-5 (δC 139.6), C-9 (δC 49.9) and C-10 (δC
36.5) (Figure S7, S8). The presence of carbonyl moiety at C-22 (at δC 214.9), was confirmed by
its HMBC correlations with methyl at δH 1.10 (3H, s, Me-21) and methylene protons at δH 2.36,
2.43 (m, H-23α, β). The esterification at C-3 has been confirmed from the correlations in HMBC
between the C=O (at δC 173.2 ppm) and H-4 protons at δH 2.31(m, H-4α, β) (Figure S7, S8). The
H-4 protons at δH 2.31 also showed HMBC correlations with C-3 (at δC 73.5) and C-5 (at δC 139.6).
The linkage of fatty acid chain was fixed at C-3 position further supported by downfield shift
of its proton at δH 4.60 (m) (Lee et al. 2014). From all the above evidences mentioned, the
structure of compound (1) was established as a new sterol 22-ketocholesteryl palmitate.
The other known compounds were identified as, 24-methylenecycloartanol (2) (Barla
et al. 2006), cycloartanol (3) (Khuong-Huu et al. 1975; Barla et al. 2006), stigmasterol (4) (Wu
et al. 1998) and linoleic acid (5) (Kamurthy et al. 2015) using comparison of NMR data with
previously published reports.
3. Experimental
3.1. General
The 1H, 13C-NMR, DEPT, HSQC, HMBC and COSY spectra were recorded on a Bruker Avance
600 NMR. The gas chromatography/mass spectrometry (GC/MS) analyses were conducted
using a Shimadzu QP 2010 using a DB-5 (J&W Scientific, Folsom, CA, USA) capillary column
(30 m × 0.25 mm i.d., 0.25 μm thickness).The mass spectrometry was performed on a Q-TOF
triple-quadrupole mass spectrometer equipped with ESI source (Micromass, Manchester,
UK) software Masslynx v4.1.The data acquisition was performed using negative ion mode
over a mass range of m/z 100–1000.
3.2. Plant material
The rhizomes of C. speciosus were collected from CSIR-IHBT farm. The plant material was
authenticated in-house by the taxonomist. A voucher specimen was deposited at CSIR-IHBT
Herbarium, Palampur, India (Voucher # PLP-17601).
3.3. Extraction and isolation
Air-dried rhizomes of C. speciosus (4 kg) were crushed and extracted by using a percolator
with 95% EtOH (7 times, 3 L × 24 h × 1, followed by 2.6 L × 24 h × 6) at room temperature.
The ethanolic extracts were combined and dried in a rotavapor at 40 5 °C to yield a semi-
solid residue (179.2 g). Part of this extract (170.0 g) was suspended in H2O (500 mL) and
successively extracted with CHCl3 (500 mL × 3, 50.2 g). Part of CHCl3 fraction (36.0 g) was
chromatographed over a dry silica gel (500 g, 230–400 mesh) column (4.5 × 48.2 cm) using

4
A. KUMAR ET AL.
a step gradient consisting of hexane: EtOAc (9: 1 to 10: 0, each 1 L) to afford five subfractions,
A (0.8 g), B (2.4 g), C (0.5 g), D (0.2 g), E (0.32 g), respectively. Sub fraction B (2.4 g) was chro-
matographed over dry silica gel (100.2 g, 230–400 mesh) column (3 × 32.6 cm) using an
isocratic solvent system consisting of EtOAc: hexane (5: 95, 3L, each fraction 50 mL) to afford
five sub fractions (B1-B5). Sub fraction B1 (Fr No. 24–31, 40.2 mg) was chromatographed over
dry silica gel (20.2 g, 230–400 mesh) column (1.8 × 18 cm) using isocratic solvent system
consisting of EtOAc: hexane (2: 98, 500 mL, each fraction 25 mL) to afford a total 30 fractions.
Fr·No.16–24, yielded compound 1 (4.2 mg). Sub fraction B3 (Fr No. 32–38, 98.2 mg) was chro-
matographed over RP-18 column (1.8 × 18 cm) using isocratic solvent system consisting
H2O: methanol (20: 80 each fraction 25 mL) and total 20 fractions were collected. Fraction
No. 6–8 after washing with acetone afforded compound 2 (12.2 mg). Fr. No. 15–20 on crys-
tallisation yielded compound 3 (5.0 mg). The subfraction B5 (510.0 mg) was chromato-
graphed over RP-18 column (2.3 × 25 cm) using isocratic solvent system consisting H2O:
methanol (30: 70 each fraction 50 mL), total 45 fractions were collected. Fraction No.1–12
dried in a rotavapor and after washing with acetone afforded compound 4 (18.6 mg). Fraction
No. 35–41 on crystallisation gave compound 5 (15.3 mg) Figure 1.
3.4. Hydrolysis of Compound 1 and GC-MS analysis
Compound 1 (2 mg) was heated under reflux with 5% methanolic KOH (1 mL) for 3 h. The
reaction product was diluted with water (10 mL) and extracted with chloroform (10 mL × 2).
The chloroform layer was dried over anhydrous Na2SO4, and the residue after evaporation
was subjected to GC-MS analysis and was identified as methyl palmitate by comparison of
RI and mass fragmentation with literature (Adams 2007; Agnihotri et al. 2009).
22-Ketocholesteryl palmitate (1): white solid.1H NMR (600 MHz, CDCl3) δH:1.13 (m, H-1α),
1.84c (H-1β), 1.85c (H-2α), 1.56c (H-2β), 4.60 (m, H-3), 2.31 (m, H-4α, β), 5.36 (m,H-6), 1.52c
(H-7α), 1.95c (H-7β), 1.43c (H-8β), 0.97 (m, H-9α), 1.54c (H-11α), 1.45c (H-11β), 1.27c (H-12α),
1.94c (H-12β), 1.04c (H-14α), 1.56c (H-15α), 1.07c (H-15β), 1.67 (m, H-16α), 1.18c (H-16β), 1.60c
(H-17α), 0.70 (s, Me-18), 1.02 (s, Me-19), 2.51 (m,H-20), 1.10 (d, J = 6.8, Me-21), 2.36, 2.43 (m,
H-23α, β), 1.43c (H-24 α, β),1.57c (H-25), 0.88c (m, Me-26), 0.89c (m, Me-27). Fatty acid chain:
2.26 (t, J = 3.0, H-2′α, β), 1.60c (H-3′α, β), 1.22–1.32c (H-4′-13′,CH2 chain),1.25c (H-14′α, β), 1.23c
(H-15′α, β), 0.87c (m, Me-16′).13C NMR (150 MHz, CDCl3) δC: 36.9 (C-1), 27.7 (C-2), 73.5 (C-3),
38.0 (C-4), 139.6 (C-5), 122.3 (C-6), 31.7 (C-7), 31.8 (C-8), 49.9 (C-9), 36.5 (C-10), 20.9 (C-11),
39.5 (C-12), 42.4 (C-13), 55.9 (C-14), 24.4 (C-15), 27.4 (C-16), 52.0 (C-17), 12.0 (C-18), 19.2 (C-19),
49.4 (C-20) 16.5 (C-21), 214.9 (C-22), 39.5 (C-23), 32.3 (C-24), 27.6 (C-25), 22.3 (C-26), 22.3
(C-27). Fatty acid chain: 173.2 (C-1′), 34.6 (C-2′), 25.0 (C-3′), 29.0–29.6 (C-4′-13′), 31.9 (C-14′),
22.6 (C-15′), 14.0 (C-16′). HRESIMS: 637.5552 ([M − H]−(Calcd for C43H73O3,637.5560). (Note: c
represents overlapped signal, assignments were done by 2D experiments.)
4. Conclusion
Western Himalayan flora is full with novel skeletons. Recently our group had introduced two
new skeletons termioic acid A (Agnihotri et al. 2014), 14R, 17S, 20R-lupan-3-one (Thakur &
Agnihotri 2016) and new rare molecule (Walia et al. 2016) from this region. In this study, the
isolation of 22-ketocholesteryl palmitate (1) from C. speciosus is the first report of a 22-oxo-
sterol derivative from plant source.

NATURAL PRODUCT RESEARCH
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Supporting material
All spectral data associated with this article are provided as supplementary file.
Acknowledgment
The authors gratefully acknowledge Dr. Sanjay Kumar, Director, CSIR-IHBT Palampur (HP), India, for
continuous encouragement and for providing the needed facilities during the course of the study.
Thanks are also due to UGC for awarding senior research fellowship to Mr. Ashish Kumar. Authors are
also grateful to CSIR, New Delhi for funding the BSC-0106 project under which the work was carried out.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Adams RP. 2007. Identification of essential oil components by gas chromatography/mass spectroscopy.
Carol Stream: Allured Publishing Corporation.
Agnihotri VK, ElSohly HN, SmillieTJ, Khan IA, Walker LA. 2009. Constituents of Leonotis leonurus flowering
tops. Phytochem Lett. 2:103–105.
Agnihotri VK, Thakur S, Pathania V, Chand G. 2014. A new dihomosesquiterpene, termioic acid A, from
Curcuma aromatica. Chem Nat Compd. 50: 665-668.
Al-Attas AA, El-Shaer NS, Mohamed GA, Ibrahim SR, Esmat A. 2015. Anti-inflammatory sesquiterpenes
from Costus speciosus rhizomes. J Ethnopharmacol. 176:365–374.
Augustin MM, Ruzicka DR, Shukla AK, Augustin JM, Starks CM, O’Neil-Johnson M, McKain MR, Evans
BS, et al. 2015. Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of
verazine in Sf9 cells. Plant J. 82:991–1003.
Barla A, Birman H, Kultur S, Oksuz S. 2006. Secondary metabolites from Euphorbia helioscopia and their
vasodepressor activity. Turk J Chem. 30:325–332.
Deni, B., 2008. Encyclopaedia of herbs. The Royal Horticulture Society. London: Dorling Kindersley.
Gupta RK. 2010. Medicinal and aromatic plants. New Delhi: CBS Publishers and Distributors.
Kamurthy H, Dontha S, Vedula A. 2015. Phytochemical screening of isolated compounds from Nymphaea
nouchali Burm.f. flowers. European J Med Plants. 9:1–12.
Khuong-Huu F, Sangare M, Chari V, Bekaert A, Barbier M, Lukascs G. 1975. Carbon-13 nuclear magnetic
resonance spectral analysis of cycloartanol and related compounds. Tetrahedron Lett. 16:1787–1790.
Lee NK, Shin HJ, Kim WS, Lee JT, Park CK. 2014. Studies on the chemical constituents of the New Zealand
Deer Velvet Antler Cervus elaphus var. scoticus-(I). Nat prod Sci 20:160-169.
Plouguerné E, Kikuchi H, Oshima Y, Deslandes E, Stiger-Pouvreau V. 2006. Isolation of Cholest-5-en-
3-ol formate from the red alga Grateloupia turuturu Yamada and its chemotaxonomic significance.
Biochem Syst Ecol. 34:714–717.
Selim S, Al Jaouni S. 2016. Anti-inflammatory, antioxidant and anti-angiogenic activities of diosgenin
isolated from traditional medicinal plant, Costus speciosus (Koen ex. Retz.) Sm. Nat Prod Res. 30:1830–
1833.
Srivastava S, Singh P, Mishra1 G, Jha KK, Khosa RL. 2011. Costus speciosus (Keukand): a review. Der
Pharmacia Sinica 2:118-128.
Thakur S, Agnihotri VK. 2016. 14R, 17S, 20R-lupan-3-one, assignment of a new triterpene structure from
Commiphora wightii. Magn Reson Chem. 54:400–402.
Walia M, Kumar D, Kumar P, Singh B, Padwad YS, Agnihotri VK. 2016. Cytotoxic new nortriterpene from
roots of Potentilla atrosanguinea var. argyrophylla and its UPLC quantification. Planta Med Int Open.
3:e47–e50. doi:10.1055/s-0042-109070.
Wu LJ, Xiang T, Hou BL, Liang W, YiS Zhou XC. 1998. Chemical constituents from fruits of Ligustrum
lucidum. Acta Botanica Sinica. 1:83–87.