Steroids 175 (2021) 108913

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Steroids
journal homepage: www.elsevier.com/locate/steroids

Review

Two new biologically active steroids from Costus lucanusianus (Costaceae)

Adesegun O. Onanuga *, Ganiyat K. Oloyede
Department of Chemistry, University of Ibadan, Ibadan, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: A phytochemical investigation of the leaf extract of Costus lucanusianus J. Braun & K. Schum (Family Costaceae) a
Costus lucanusianus J. Braun & K. Schum tropical African medicinal plant known for curing several infectious diseases such as venereal disease, cough and
Urinary tract infection urinary tract infection led to the isolation of two new steroids. The identification of these isolates was achieved
3,27-dihydroxy-1-methoxy-22-cholest-5-enone
by modern spectroscopic methods, including 2D NMR. The in vitro antimicrobial activity and Minimum Inhi­
bition Concentration (MIC) values of the isolated compounds against six bacterial and four fungi strains were
evaluated.
Compound Xp named 3,27-dihydroxy-1-methoxy-22-cholest-5-enone and compound 1 named β-sitosterol-3-
O-β-D-3-deoxyxylo-4-hydroxy4,5-dimethyl-pent-2-one displayed broad antimicrobial activity at concentration
12.5 µg/mL-100 µg/mL. Compound Xp displayed MIC value 25.0 µg/mL against tested micro-organisms except
for P. notatum and R. stolonifer which showed no prominent growth. Compound 1 was insufficient to determine
the MIC value.
This present study may be helpful in discovering new chemical groups of antimicrobial compounds that could
be useful as an agent against infectious diseases.

1. Introduction others [5]. Pharmacological activities of crude extracts such as tocolytic

property [6], antimicrobial [7] and antidiarrhoeal [8] have been re­
Infectious diseases are known globally to account for 13.4 million ported. However, little is known about the chemical components of
deaths per year [1]. Infectious diseases have huge impact in emerging C. lucanusianus [9]. This necessitated the isolation of bioactive compo­
countries due to increasing drug-resistant infections and relative un­ nents from the plant C. lucanusianus. Costus genus has been reported to
availability of drugs [2]. The resistance of microbes to antimicrobial be rich in sapogenins, oxalates, steroidal saponins, furan and their de­
agents has brought about a great challenge in the treatment of infectious rivatives, and starches [10].
diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the
major persistent human infections. Alteration of the molecular structure 2. Experimental
of an antibiotics, reduction of the intracellular drug concentration by
expressing efflux pump, enzymatic inactivation of antibiotics and 2.1. Chemical and reagents
modification of the antibiotic targets are the known mechanisms of the
resistance of bacteria to antibiotics. The need for new small molecules Chemicals and reagents used were of analytical grades and were
that are able to counteract the mechanisms underlying antibiotic resis­ supplied by the department chemical store.
tance is urgently needed. Thiazole and its benzofused derivative have
been reported to inhibit the mechanisms underlying antibiotic resistance
2.2. Equipment and apparatus
in the treatment of severe drug-resistant infection [3,4]. Costus lucanu­
sianus J. Braun & K. Schum (Costaceae) is known as the African spiral
UV lamp (254/366 nm), iodine vapour and acidified KMnO4 spray
flag and Spiral ginger. It is also usually referred to as monkey sugar cane
were used for visualising TLC plates. Quadrupole-time-of Flight (ToF)
in the South-south region of Nigeria. C. lucanusianus is known in eth­
mass spectrometer was used to determine the molecular mass. The NMR
nomedicine for its efficacy in the treatment of infectious diseases such as
spectra were acquired using Bruker Topsin 400 and 100 MHz spec­
cough, an eye drop to control filariasis, stomach problems amongst
trometer for 1H and 13C respectively. Bruker Platinum ATR Tensor 27

* Corresponding author. Tel.: +234 8033699094.

E-mail address: adesegunonas@gmail.com (A.O. Onanuga).

https://doi.org/10.1016/j.steroids.2021.108913
Received 23 April 2021; Received in revised form 9 August 2021; Accepted 25 August 2021
Available online 2 September 2021
0039-128X/© 2021 Elsevier Inc. All rights reserved.
A.O. Onanuga and G.K. Oloyede Steroids 175 (2021) 108913

FT-IR spectrophotometer was used to acquire Fourier Transform Infra- 2.8. Antifungal and antibacterial assay of isolated compounds
red (FT-IR) spectra.
2.8.1. Microbial cultures
Clinical strains tested were supplied by the pharmaceutical micro­
2.3. Plant collection and identification
biology laboratory, University of Ibadan, Ibadan. Four fungi strains used
were Penicillium notatum, Candida albicans, Aspergillus niger and Rhizopus
Samples of C. lucanusianus leaf were obtained fresh from Akobo
stolonifer while the six bacterial strains utilised were Pseudomonas aer­
Ibadan, Oyo state, South-west, Nigeria (at a geographical coordinate of
uginosa, Salmonellae typhi Staphylococcus aureus, Bacillus subtilis, Escher­
7◦ 25! 45.4′′ N 3◦ 56! 10.9′′ E; altitude 830 ft.) on 14th October 2015.
ichia coli and Klebsiella pneumoniae.
The taxonomic identification of the plant materials was confirmed by a
senior plant taxonomist, Mr L.T Soyewo of Forestry Research Institute of
2.8.2. Antimicrobial activity method
Nigeria (FRIN) herbarium, Ibadan. Voucher specimens (FH110048)
Stock solutions of compound Xp and 1 were prepared by dissolving 1
were deposited at the FRIN herbarium. The collected leaves were air-
mg each in 10 mL of methanol for proper dilution to obtain a final
dried for two weeks under shade. These were ground and kept in
concentration of 100 µg/mL [11]. Four different concentrations of the
airtight containers until use.
stock solutions (12.5 µg/mL-100 µg/mL) were obtained by double-fold
serial dilution.
2.4. Extraction procedures
2.8.3. Preparation of bacterial inoculum
Ground leaves (1.3 kg) was extracted with hexane for 22 h in soxhlet A loop full of each glycerol stock-culture was inoculated into a tube
apparatus. Further extraction of the marcs obtained with methanol for containing 5 mL each of sterile nutrient broth (SNB). This was vortexed
30 h was done until the solvent in the thimble became clear. The cautiously well and thereafter incubated at 37 ◦ C for 24 h. Stand­
resulting methanol portion was partitioned with ethyl acetate by solvent ardisation of the bacterial suspension (inoculum) was performed with
fractionation method. Solvents were evaporated from the extracts with sterile saline water to 108 CFU/mL (turbidity = McFarland barium sul­
the aid of steam bath until almost dryness. The extracts were kept in a phate 0.5). An initial 1:100 dilution of the organisms was prepared by
desiccator over anhydrous sodium sulphate. The weights and percentage adding 0.1 mL of the overnight culture to sterile distilled water (9.9 mL).
yields (w/w) of the extracts were determined. Subsequently, 0.2 mL of the solution of the organism was taken and
added into sterile nutrient agar (20 mL) which was at 45 ◦ C [12] with
modification.
2.5. Column chromatography separation (CCS) of the leaf ethyl acetate
fraction 2.8.4. Antibacterial assay (Agar well diffusion method)
The organisms and the mixture of sterile nutrient agar were carefully
Ethyl acetate fraction (9 g) was purified on silica gel (60–200 mesh, transferred into sterile petri dishes and were allowed to solidify for 60
270 g, 32.8 mm × 60 mm) using varying concentration of ethyl acetate min. 30 µL of compounds Xp and 1 solution in methanol, gentamicin and
in hexane. methanol were introduced into the wells (8 mm diameter hole pierced in
A total of 178 fractions of 200 mL each were collected from the the agar at 25 mm apart from one another using sterilized cork borer).
column and these were mixed together into 17 sub-fractions following These were permitted to diffuse well for 60 min at room temperature.
the TLC pattern. The sub-fraction 17 (yield = 80 mg) was purified with a Subsequently, the plates were incubated for 24 h at 37 ◦ C. Following this
second CCS. A solid (yield = 55 mg) was obtained at 80% ethyl acetate was the observation of bacterial growth zones. Gentamicin (10 µg/mL)
in hexane. Ethyl acetate: methanol 5:1 and 7:1; diethyl ether: methanol was utilised as a positive control while the negative control was meth­
4:1were the solvents mixtures used for the TLC analysis. anol. The bacterial inhibition zone diameter (IZD) were measured with a
Ethyl acetate 5: methanol 1which gave the best separation on TLC metre ruler in mm. Tests were carried out in triplicate.
plates was further used to purify the solid obtained. This was followed
by recrystallization from hot methanol to obtain an ash coloured solid 2.8.5. Antifungal assay: Preparation of inoculum
which was labelled compound 1. The melting point and yield of com­ The fungi strains taken from the stock were subcultured on to Sab­
pound 1 were determined. ourand dextrose agar (SDA). The mixture was incubated at 35 ◦ C for
three days. The yeast spores obtained were suspended in sterile distilled
water (5 mL) to obtain 105cells/mL. Dilution of the organisms to 1:100
2.6. Column chromatography separation (CCS) of the leaf hexane extract
was carried out and 0.2 mL of 1:100 dilution of the adjusted inoculum
was taken and spread over the agar using a sterile spreader [13].
Hexane extract (38.55 g) was purified on silica gel (Merk 60–200
mesh, 1200 g, 45.5 mm × 90 mm) by solvent gradient method using
different concentration of ethyl acetate in hexane and100% n-hexane. A 2.8.5.1. Surface plate method. The prepared sterile (SDA) (62 g/L) was
total of 190 fractions of 200 mL each were collected from the column. left to solidify for 45 min in sterile plates in triplicate. The antifungal
Similar fractions following the TLC patterns as visualised in iodine activity of the compounds was determined by using 30 µL of the each
vapour, UV lamp (254 and 365 nm) and KMnO4 spray (heated in oven at concentration (12.5 µg/mL − 100 µg/mL). The experiment was per­
100 ◦ C for 5 min) were mixed together to form 18 sub-fractions. Frac­ formed in triplicate on (SDA) impregnated with clinical fungal strains.
tions (86–101) obtained from the main column was a mixture of need- Wells were made inside the set plates using 8 mm diameter sterile cork
like crystals and powdered solid. This mixture was purified repeatedly borer. Different concentrations of the compounds, and the controls
with column chromatography separation utilising different concentra­ (tioconazole 70%) and methanol were poured into the wells and there
tion of ethyl acetate in hexane to afford a colourless amorphous powder was perfect diffusion into the agar for 120 min. Thereafter, the incu­
(20 mg) labelled Xp. The melting point of Xp was determined. bation of the plates uprightly for 48 h at 28 ◦ C took place. The inhibition
zone diameter (IZD) in mm was measured [14].

2.7. Structural elucidation of compounds Xp and 1. 2.8.6. Minimum inhibitory concentration (MIC)
Various concentrations of compounds 1 and Xp were made by dis­
The secondary metabolites Xp and 1isolated and purified were solving them in dimethylsulphoxide. Agar dilution method (micro-
structurally elucidated with the help of spectroscopic techniques.

2
A.O. Onanuga and G.K. Oloyede Steroids 175 (2021) 108913

dilution technique) was utilised for determining the MIC. Prepared 3.2. Elucidation of compound Xp
sterilized agar was allowed to cool to 45 ◦ C, and 18 mL each was thor­
oughly mixed with different concentrations (2 mL each) of the com­ Compound Xp was an amorphous white powder of (yield:20 mg) and
pounds. The mixture was permitted to uniformly spread in a 96-well melting point of 129 ◦ C. IR (KBr) cm− 1 spectrum (Appendix B1) revealed
microplate after which the plate was kept in an oven for 24 h at 37 ◦ C. absorption peaks at 3500 cm− 1 and 1655 cm− 1 (–OH, -C– – C-respec­
Thereafter, the micro-organisms were applied on the surface. The tively); 1021 cm− 1(C–O). Many strong bands between 875 and 1350
experiment was done in triplicate and the MIC of the compounds in cm− 1 which are typical of the spiroketal side chain.
1
millimetre was determined [15]. H NMR (400 MHz, cdcl3) (Appendix B2); δ H 5.20 (dd, J = 8.0 Hz,
H-6), 3.42–3.49 (H-1, 3 and 26), 1.6–0.9 (m, H-methylene, methyl). The
13
C NMR spectrum (Appendix B3) broadband signals were resolved with
2.9. Statistical analysis DEPT 135 experiment (Appendix B4) into four quaternary, five methyl,
nine methine and ten methylene carbon atoms (Table B1). The APCI-
All data were subjected to statistical analysis which was determined ToF-MS/MS spectrum (Appendix B5) of Xp revealed base peak at m/z
by making use of one way ANOVA. The differences between the data 397.3070 [C27H41O2] and a double bond equivalent 5.5. [M+] peak at
were considered significant at P ≤ 0.05. m/z 446.2996.
Antimicrobial activity of isolated compounds
3. Results Compounds 1 and Xp displayed broad antimicrobial activity against
tested bacterial strains at 12.5 µg/mL-100 µg/mL as shown in
The percentage yields of Costus lucanusianus leaf hexane extract and (Table C1).
ethyl acetate fraction were 3.40 and 1.60 respectively. Compound Xp MIC result as shown in (Table C2).

4. Discussion
3.1. Elucidation of compound 1
4.1. 4.1: Elucidation of compound 1
Compound 1was an amorphous ash-coloured solid of (yield:25 mg).
1
The melting point was 235–236 ◦ C. H NMR (DMSO‑d6, 400 MHz) of compound 1 displayed several
IR (KBr) cm− 1 spectrum (Appendix A1): revealed absorption peaks at signals of (–CH2, –CH3 protons) in the aliphatic area of the spectrum
3420 cm− 1(–OH), 2910 and 2850 cm− 1 (–CH stretching of CH3 and CH2 within the range (δH 0.6–1.1 ppm). Proton equivalent to H-3 of the
groups), 1021 cm− 1 (C–O) and 1640 cm− 1 (C– – C). aglycone in compound 1 which appeared as a multiplet at δH 3.4 ppm
1
H NMR (DMSO‑d6, 400 MHz) (Appendix A2): 4.1(bs, H-1′′ of the corroborates that reported for H-3 of sterol moiety which usually ap­
sugar moiety), 3.6 (s, OH-of sugar moiety), 3.4 (m, H-3 of the aglycone), pears at δH 3.4 ppm as a multiplet [16]. The H-1 of sugar moiety which
3.3 (m, H-2′ of the sugar moiety), 3.0 (m, H-3 of the sugar moiety), 2.1 also usually appear as a multiplet at δH 4.1 [17] appeared as a broad
(s, CH3-of the acetyl group), 1.1–0.6 (m, H-methylene, methyl). 13C singlet of the sugar moiety in compound 1. Angular methyl protons at δ
NMR (DMSO‑d6, 100 MHz) (Appendix A3): The broadband displayed six 0.8 and δ 0.9 harmonised to C-18 and C-19 protons respectively.
signals: δC 207 (C– – O), 79 (C-3), 56 (CH2), 49 (CH), 31(CH3) and 18 Out of the 38 carbon atoms revealed by the Dept 135 data, 29 car­
(CH3). These signals were further resolved with DEPT 135 (Appendix bons atoms were similar to those reported values for β-sitosterol were 11
A4) to contain 38 carbon atoms (Table A1). ESI-ToF/MS/MS in the methylenes, 6 methyls, 9 methines and 3 quaternary carbons (Table A1)
positive mode (Appendix A5): revealed the base peak at m/z 397 [18,19]. Other prominent peaks at δC 102 and 79.0 were observed in the
[C29H49]+ having five degree of unsaturation (DBE = 5) and [M+] peak DEPT 135 data. These were assigned to the oxygenated carbon C-1 of the
at m/z 658. xylose and C-3 of sitosterol linked to oxygen atom respectively. The
presence of a sugar moiety was established by a carbon chemical shift at
Table A1 δC 102 and an anomeric proton at δH 4.1.
The dept 135 data of Compound 1. The δC 19 and δC 12 were allotted to the angular methyl carbons of C-
Carbon no δ value Assignment Carbon no δ value Assignment
19 and C-18 respectively of sitosterol [20]. The unsaturation assigned
between C-5 and C-6 of the aglycone (sitosterol) was substantiated by
1 36.60 CH2 16 28.64 CH2
the correlation noticed between carbon at δC 140 and proton at δH 0.9 in
2 29.21 CH2 17 56.64 CH
3 79.00 CH 18 12.00 CH3
4 41.60 CH2 19 19.00 CH3
5 140.00 Cq 20 33.28 CH Table B1
6 121.00 CH 21 18.40 CH3 The dept 135 data of methyl, methylene, methine and quaternary carbon of
7 31.00 CH2 22 33.00 CH2 compound Xp*.
8 31.08 CH 23 25.38 CH2
9 50.01 CH 24 45.61 CH Carbon No δ value Assignment Carbon No δ value Assignment
10 363.12 Cq 25 28.64 CH 1 72.50 CH 15 24.12 CH2
11 20.50 CH2 26 18.73 CH3 2 34.88 CH2 16 26.54 CH2
12 38.20 CH2 27 18.58 CH3 3 69.73 CH 17 55.00 CH
13 41.60 Cq 28 22.06 CH2 4 38.00 CH2 18 19.00 CH3
14 55.80 CH 29 12.20 CH3 5 141.00 Cq 19 12.07 CH3
15 23.00 CH2 C-1A 12.00 CH3 6 121.70 CH 20 31.00 CH
C-11 102.00 CH C-2A 207.00 C–
–O 7 32.00 CH2 21 17.06 CH3
C-21 77.00 CH C-3A 56.40 CH2 8 31.05 CH 22 206.97 C–
–O
C-31 49.00 CH C-4A 78.00 Cq 9 48.98 CH 23 33.00 CH2
C-41 71.00 CH C-5A 34.00 CH 10 36.40 Cq 24 30.27 CH2
C-51 65.80 CH2 C-6A 12.00 CH3 11 22.05 CH2 25 34.44 CH
C-7A 23.10 CH3 12 39.37 CH2 26 13.85 CH3
13 13 43.83 Cq 27 67.23 CH2
C Spectrum (DMSO‑d6) recorded on Bruker Avance III 400 at 100.0 MHz. Cq =
14 56.50 CH 28 56.96 CH3
quartenary.
carbon. C-1-C-29 = Carbon atoms of Sitosterol, C-11-C-51 = Carbon atoms of 13
C spectrum recorded on Bruker Topsin III 400 at 100.0 MHz in CDCL3. Cq =
xylose, C-1A-C-7A = Substituents on the xylose unit. quaternary carbon.

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A.O. Onanuga and G.K. Oloyede Steroids 175 (2021) 108913

Table C1
Antimicrobial activity of compounds Xp and 1.
Concentration (μg/mL) S. aureus E. coli B. subtilis P. aeruginosa S. typhi K. pneumonia C. albicans A. niger P. notatum R. stolonifer

Compound 1
100 27.3 ± 26.7 + 23.3 + 23.3 + 1.31 20.7 + 18.7 + 1.31 20.0 + 0.00 18.7 + 18.0 + 0.0 18.7 + 1.31
1.31 1.31 1.31 1.31 0.00
50 23.7 ± 22.7 + 20.0 + 21 + 1.13 18.0 + 0 16.7 + 1.31 18.0 + 0.00 16.0 + 0.0 16.0 + 0.0 17.0 + 1.60
0.65 1.31 2.26
25 20.7 ± 18.7 + 16.7 + 18.7 + 1.31 16.7 + 14.7 + 1.31 16.0 + 0.00 14.0 + 0.0 15.3 + 1.31 14.0 + 0.00
1.31 0.65 1.31 1.31
12.5 18.7 ± 15.3 + 14.0 + 0.0 16.7 + 1.31 15.0 + 12.7 + 1.31 14.0 + 0.0 12.0 + 0.0 13.3 + 1.31 12.0 + 0.00
1.31 1.31 1.13

COMPOUND Xp
100 27.3 ± 18.7 + 24.0 + 23.3 + 1.31 18.7 + 20.7 + 1.31 20.0 + 0.0 18 + 0.0 18.0 + 0.00 18.7 + 1.31
1.31 1.31 0.00 1.31
50 24.3 ± 16.7 + 20.7 + 20.0 + 1.13 16.7 + 18.3 + 0.65 18 + 0.0 16 + 0.0 16.0 + 0.00 14.0 + 0.00
0.65 1.31 1.31 1.31
25 20.7 ± 14.0 + 17.3 + 17.7 + 0.65 14.0 + 0.0 16.3 + 0.65 16 + 0.0 14 + 0.0 14.0 + 0.0 12.0 + 0.00
0.65 0.00 1.31
12.5 17.7 ± 12.7 + 14.7 + 15.3 + 0.65 12.0 + 0.0 14.3 + 0.65 14.0 + 0.0 12 + 0.0 13.3 + 1.31 10.0 + 0.00
0.65 1.31 1.31
− ve Methanol – – – – – – – – – –
+ve Gentamicin (10 µg/ 38 38 38 38 38
mL)
Tioconazole (70%) 28 26 28 2

Table C2
Minimum inhibitory concentration of compound Xp.
Concentration (μg/mL) S. aureus E. coli B. subtilis P. aeruginosa S. typhi K. pneumonia C. albicans A. niger P. notatum R. stolonifer

50 − − − − − − − − − −
25 − − − − − − − − ± ±
12.5 − + ± ± ± + + + + +
6.25 − + + + + + + + + +
3.125 + + + + + + + + + +

the HMBC spectrum (Appendix A6). The existence of an olefin absorp­ Additional detected ions are listed in Table A2. These ions were similar
tion bond was also substantiated by IR band at 1640 cm− 1. The full to some of the fragments observed in 3-β-D-glycopyranosyl-β-sitosterol
structural elucidation was established by comparison with the literature [22]. The peaks at m/z 396 and 397 are typical of β-sitosterol [16,23].
and the data obtained from mass spectroscopy experiments (Appendix Ion at m/z 299 which has been reported as a specific fragment for sterol
A5). with saturated side chain [24] further supported the fact that the agly­
All the proton signals were correlated to their respective carbons cone was not stigmasterol. Few fragment ions detected by the ESI-ToF/
with the help of the HMQC experiment. At the side chain, the attach­ MS/MS could have been due to D-xylose which is known for its existence
ment of a methyl group to the quaternary carbon (C– – O) was established in pyranosic form in aqueous solution.
by the correlation noticed in the HMBC spectrum between protons at δH The proposed pathway for the formation of m/z 468 was the frag­
2.1 (CH3) and a quaternary carbon at δC 207 and also the cross peak mentation of the xylose at C-31-C-41 bond to give a molecule (− 486),
noticed between δC 31 and δH 2.1 in the HMQC spectrum (Appendix A7). then loss of water molecule (− 18) to give m/z 468 (Fig. 1).
There was a broad intense singlet (which indicated the existence of a The most characteristic peak of steroidal sapogenin is at m/z 139.
β-xyloside unit) of anomeric proton at δH 4.1. The protons of sugar Also, in the fragmentation of steroids the loss of the methyl group is the
moiety except for the H-1 usually appear at about δH 3.2 [21]. The first point of interest. The loss CH3 (m/z − 15) from sitosterol (m/z =
downfield chemical shift of the protons of the sugar moiety in compound 414) gave m/z 399. Molecular formula for compound 1 was established
1 could have been due to the existence of the acetyl group in the sugar to be C41H70O6. Acylated sterol glycosides are known to occur naturally
unit. In the HMQC where the carbon at δC 49 revealed a cross-peak with in plant tissues and the acylation usually assumed to be at C-6. However
proton at δH 3.2 of the sugar moiety, this suggested the point of linkage C-4 acylation was also reported by [25]. Herein, we reported the C-3
of the substituent to the sugar [18] and also the presence of 3-deoxy acylation of deoxy-xylose.
xylose. The C–C linkage at δC 49 was established by the correlation Sitosterols have been reported to undergo oxidation processes to
observed in the HMBC between carbon at δC 49 and protons at δH 4.1 form sitosterol oxidized products. Different derivatives of sitosterol that
and δH 3.0, δ 3.2. have been isolated include β-sitosterol diglucosyl caprate, β-sitosterol-3-
The sugar unit point of attachment to the aglycone was established as O-butyl, 7-keto sitosterol, β-sitosterol glucoside-3-O-hexacosanoicate,
1–3 with the help of downfield methine carbon signal of sugar at δC 102 β-sitosterol-3-O-β-D-xylopyranoside and 7-O-β-D-fucosyl-3-oxo-4-en
and the C-3 downfield chemical shift of (δC 79) as opposed to the δC 71 clerosterol [21,25,26,27]. However, a new addition to the family of
for C-3 in β-sitosterol [20] and other connectivities noticed in the HMBC steroids having acylated 3-deoxy-xylose is hereby reported. Compound
spectrum. 1 was trivally named β-sitosterol-3-O-β-D-3-deoxyxylo-4-hydroxy4,5-
The ESI-ToF/MS/MS in the positive mode showed the utmost intense dimethyl-pent-2-one (Fig. 2).
peak at m/z 397 which was equivalent to [C29H49]+ having five degree
of unsaturation (DBE = 5):- four rings and an olefinic bond (This sug­ 4.2. Elucidation of compound Xp
gested the aglycone part to be sitosterol). The protonated molecular ion
[M+] peak was at m/z 658. A precusor ion was also observed at m/z 606. Few signals were recognisable in the 1H NMR spectrum as a result of

4
A.O. Onanuga and G.K. Oloyede Steroids 175 (2021) 108913

Fig. 1. Fragmentation pattern of β-sitosterol-3-O-β-D-3-deoxyxylo-4-hydroxy4,5-dimethyl-pent-2-one.

extensive inter-proton coupling. For the signals (>3ppm) such as oxy spectrum and the correlation detected between carbon at δ 141 and
substituted methylene and methine resonances, olefinic, and the angular proton δ 0.9 in the HMBC spectrum. Correlations were also noticed in
and secondary methyl signals are characteristic signals in the 1H NMR of the HMBC spectrum between δC 207 and δH 2.1; δC 31 and δH 1.9, 2.1
steroidal sapogenin because CH3 and CH2 resonances were observed as and 2.3 while cross-peaks were also noticed in the HMQC spectrum
multiplets (overlapping of signals) [28]. between δC 31 and δH H 2.1; δH 0.9 and δC 19. All carbon and proton
Four methyl singlets were observed between δH 0.6 and δH 1.56 in assignments were finally made with COSY, HMBC, HMQC and NOESY
the proton spectrum of compound Xp. Various connectivities in Xp were experiment. The mass fragmentation pattern of Xp observed in the APCI-
determined through HMBC experiment. H-1, 3 and 26 appeared as ToF-MS/MS clearly indicated the presence of spirostanol type of skel­
multiplet within the range of δ 3.42–3.49. eton [28].
The broadband 13C NMR spectrum suggested the skeletal type of In the APCI-ToF-MS/MS spectrum of compound Xp, there was a loss
compound Xp (spirostanol type). The parent skeleton of compound Xp of fragment m/z = 31 which resulted into m/z 415.3174, and this sup­
was established with C-22 multiplicity and chemical shift values. The C- ported the existence of a methoxy group in the molecule. An absorption
22 of compound Xp was of keto group and of chemical shift value of δ band at 1020 cm-1in the infra-red spectrum further confirmed the
206.97 which was contrary to the chemical shift values δ 34.8–35.4 presence of C–O–C bond.
reported for the 22-deoxy- spiroidal sapogenin of 16-hydroxycholestane In APCI-ToF/MS/MS, the main fragment ions were derived from the
skeleton type [28]. The presence of four methyl resonances is the loss of CH3OH (m/z = 31) followed by the protonation and formation of
common features of all steroidal sapogenin however, five methyl reso­ positive charge on the oxygen at C-16 to produce m/z 416. Subse­
nances may occur occasionally [28] as in compound Xp (two tertiary quently, the transfer of electron pair from C-16 to oxygen brought about
methyls and three secondary methyls). the breaking up of C-16-O bond. Carbonyl at C-21 was produced by the
The most commonly encountered unsaturation in spiroidal sapo­ tautomerisation due to the presence of an enol. The dissociation of the
genin is the double bond at C-5 which resulted into the display of C-5 intermediate took place in three pathways (Fig. 3).
and C-6 at δ 141.0 (C) and δ 121.9 (CH) respectively. This can be sub­ In pathway A (main), there was the breaking up of C-17-C-20 bond.
stantiated by the absorption observed at 5.28 ppm (dd) for the H-6 This resulted into the production of m/z 271.20.
signal. The unsaturation was also further definite by the cross-peak In pathway B (minor), there was transference of hydrogen from C-23
noticed between carbon at δ 121 and proton δ 5.28 in the HMQC to C-20 after which there was the breaking up of C-20-C-22 bond. Lastly,

5
A.O. Onanuga and G.K. Oloyede Steroids 175 (2021) 108913

Fig. 2. Structure of β-sitosterol-3-O-β-D-3-deoxyxylo-4-hydroxy4,5-dimethyl-pent-2-one.

Fig. 3. Fragmentation pathways of aglycone of steroid saponin (Xp) using APCI-QqTOF-MS/MS.

the formation of m/z 283.24 was an outcome of the H2O molecule methoxy-22-cholest-5-enone (Fig. 4).
removed.
In pathway C, there was a sequential removal of two molecules of
4.3. Antimicrobial activity of isolated compounds
water to generate ions at m/z 397.31 and 379.29 respectively.
From the above, the molecular weight of Xp was found to be 445
Antimicrobial activity of different concentrations (12.5–100 μg/mL)
with molecular formula C28H45O4 and was named 3,27-dihydroxy-1-
of compounds Xp and1 was evaluated by Agar well diffusion method.

6
A.O. Onanuga and G.K. Oloyede Steroids 175 (2021) 108913

1 and Xp against E. coli, S. aureus, B. subtilis and C. albicans justifies the

use of C. lucanusianus in the treatment of infectious diseases such as
urinary tract infection, urethral discharge and venereal diseases in
ethnomedicine.
Compound Xp MIC result as shown in (Table C2) revealed that it
repressed the growth of all tested microorganisms at 25.0 µg/mL. An
exception was with P. notatum and R. stolonifer which showed no
prominent growth. Compound Xp displayed IZD against S. aureus at
6.25–50 µg/mL suggesting the high sensitivity of S. aureus to compound
Xp. Compound 1 was not sufficient to determine its MIC values.

Acknowledgement

Authors are grateful to Mr Soyewo L.T of Forestry Research Institute

Fig. 4. Structure of compound 3,27-dihydroxy-1-methoxy-22-cholest-5-enone. of Nigeria, Ibadan for the identification of the plant and Mr Festus of
Pharmaceutical Microbiology Department, University of Ibadan for
The compounds inhibited the tested microorganisms with varying helping with the carrying out the antimicrobial analyses.
sensitivity. Maximum activity was noticed at100 μg/mL of the com­
pounds. Among the bacteria, high inhibition zones were observed by Appendix A. Supplementary data
compound 1against S. aureus and E. coli while compound Xp showed
high inhibition zone against S. aureus followed by B. subtilis. This shows Supplementary data to this article can be found online at https://doi.
the high sensitivity of gram positive bacteria to compound Xp. However, org/10.1016/j.steroids.2021.108913.
the hexane crude extract from which compound Xp was obtained
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