Steroids from herbs of Reineckia carnea and their anticomplement activities

Article abstract of DOI:10.1080/14786419.2017.1423309

A new polyhydroxylated pregnane, named lβ,2β,3β,4β,5β,6β-hexolhydroxy-pregn-16-en-20-one (1), along with nine known (2–10) steroidal saponins were isolated from the whole plant of Reineckia carnea.?Structure elucidations?of all compounds were established by interpretation of their NMR spectral data, HR-ESI-MS and comparing with literatures. In addition, these compounds were evaluated with anticomplement activity. The result showed that compound 1 exhibited anticomplement effects with the CH₅₀ values of 0.043?mg/mL, but saponins (2–10) showed no inhibition. Interestingly, hydrolysis of steroidal saponins (2–10) resulted in its aglycones (2a–10a) correspondingly which showed anticomplement activity with the CH₅₀ values of 0.049–0.156?mg/mL.

Full text of DOI:10.1080/14786419.2017.1423309

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Steroids from herbs of Reineckia carnea and their
anticomplement activities
Xu Xu, Bei Wu, Yanzhi Zhan, Wenping Huang, Shilin Yang, Quan Wen & Yulin
Feng
To cite this article: Xu Xu, Bei Wu, Yanzhi Zhan, Wenping Huang, Shilin Yang, Quan Wen & Yulin
Feng (2018): Steroids from herbs of Reineckia carnea and their anticomplement activities, Natural
Product Research, DOI: 10.1080/14786419.2017.1423309
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2017.1423309
Steroids from herbs of Reineckia carnea and their
anticomplement activities
Xu Xua1, Bei Wub1, Yanzhi Zhana, Wenping Huangc, Shilin Yanga,c, Quan Wena and
Yulin Fenga,c
aJiangxi university of traditional chinese Medicine, Nanchang, china; bNanchang Insitute for Food and
c
drug control, Nanchang, Pr china; state Key laboratory of Innovative drug and efficient energy-saving
Pharmaceutical equipment, Jiangxi university of traditional chinese Medicine, Nanchang, china
ABSTRACT
ARTICLE HISTORY
received 9 November 2017
accepted 26 december 2017
A
new polyhydroxylated pregnane, named lβ,2β,3β,4β,5β,6β-
hexolhydroxy-pregn-16-en-20-one (1), along with nine known
(2–10) steroidal saponins were isolated from the whole plant of
Reineckia carnea. Structure elucidations of all compounds were
established by interpretation of their NMR spectral data, HR-ESI-
MS and comparing with literatures. In addition, these compounds
were evaluated with anticomplement activity. The result showed
that compound 1 exhibited anticomplement effects with the CH50
values of 0.043 mg/mL, but saponins (2–10) showed no inhibition.
Interestingly, hydrolysis of steroidal saponins (2–10) resulted in its
aglycones (2a–10a) correspondingly which showed anticomplement
activity with the CH50 values of 0.049–0.156 mg/mL.
KEYWORDS
Reineckia carnea;
polyhydroxylated pregnane;
steroids; anticomplement
activity
1. Introduction
Reineckia carnea (Andr.) Kunth. is a monotypic plant species in the Reineckia genus of the
Liliaceae family (Takeda et al.1961; Zhang et al. 2016). It only distributes in China and Japan
(Zhang et al. 2011). In China, it mainly distributes around Yangzi valley, and is an important
medical plant to Miao nationality for haemostasia, detoxification, relieving the inflammation,
clearing away the lung-heat, relieving cough and inhibiting Oncomelania hupensis (Han
CONTACT Quan Wen
1co-first author.
12111030025@fudan.edu.cn; Yulin Feng
fengyulin2003@126.com
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1423309.
© 2018 Informa uK limited, trading as taylor & Francis Group

2
X. XU ET AL.
et al. 2010; Xing et al. 2011; Wang, Huang et al. 2013). And modern researches have been
demonstrated that the chemical components contained in the plant were saponins with
spirostanol, cholestane, stigmastane, pregnane and ergostane type glycosides (Yang et al.
2010; Song, Zhang et al. 2015; Kanmoto et al. 1994; Zhang et al. 2007). As part of our research
project to explore more diversity bioactive leading compounds from the Miao medicine,
the chemical constituents and pharmacological studies of R. carnea were studied, and one
new polyhydroxylated pregnane lβ,2β,3β,4β,5β,6β- hexolhydroxy-pregn-16-en-20-one (1)
and nine known steroidal glycosides ophiopogonin T (2), (25S)-5β-spirostan-1β,2β,3β,4β,5β-
pentol-5-O-β-D-glucopyranoside (3),kitigenin 5-O-β-D-glucopyrano side (4), (20S,
22R)-spirost-25(27)-en-1β, 2β, 3β,4β,5β-pentaol-5-O-β-D-glucopyranoside (5), (20S,
22R)-spirost-25(27)-en-1β,3β, 4β,5β-tetraol-5-O-β-D-glucopyranoside (6), (17,20-S-trans)-5β-
pregn-16-en-1β,3β- diol-20-one 1-O-β-D-xylopyranosyl-(2→1)-[α-L-rhamnopyranosyl]-3-O-
α-L-rhamno pyranoside (7), (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol 1,16-di(β-D-glu-
copyra noside) (8), (1β,3β,16β,22S)-cholest-5-ene-1,3,16,22-tetrol 1-[O-α-L-rhamnopyranosyl-
(1→2)-β-D-glucopyranoside]16-(β-D-glucopyranoside) (9), dioscin (10) were acquired. This
paper describes the isolation and structural elucidations of the isolated compounds and
their anticomplement activities.
2. Results and discussion
Compound 1 was obtained as white amorphous powder, the HR-ESI-MS gave a pseudomo-
lecular ion at m/z 395.2067 [M − H]− that together with NMR spectroscopic data were con-
sistent with the molecular formula of C21H32O7. The 1H NMR spectrum of 1 (Figure S4) showed
two tertiary methyl signals at δH 1.45 (3H, s, H-19), 0.94 (3H, s, H-18), one methyl δH 2.24 (3H,
s) linking with carbonyl, and the presence of an olefinic proton at 6.92 (1H, dd, J = 1.8, 3.1 Hz,
H-16). According to carbon signals (Figure S5), 1 was the derivatives of C21-steroids. Moreover,
13C NMR displayed one carbonyl carbon at δC 199.6 (C-20) and two olefinic carbons at δC
147.2 (C-16) and 156.5 (C-17), which indicated the presence of an α,β-unsaturated carbonyl
group (Pan et al. 2003) (Table S1). The HMBC (Figure S8) correlations of Me-18 to C-12, C-13,
C-14 and C-17, H-16 to C-13, C-15, C-17 and C-20 were observed. The above data inferred
that C and D rings of 1 were almost consistent with those of 3β,5α,6β-trihydroxypregn-16-
en-20-one (Wang et al. 2004).
However, 13C- DEPT135 (Figure S6) displayed five hydroxy-methine at δC 67.4, 70.1, 70.7,
75.9 and 80.1, and one oxygen-quaternary carbon 79.3, and corresponding protons at δH
3.78, 3.92, 4.21, 4.11, 3.77, which indicated A ring of 1 was polyhydroxylated. In HMBC spec-
trum (Figure S8), the correlations of δH 1.45 (Me-19) to C-1, C-10 and C-9, δH 3.77 (H-1) to C-2,
C-3 and C-9, δH 4.11 (H-3) to C-1, C-4 and C-5, δH 4.21 (H-6) to C-4, C-7 and C-8 were observed.
Moreover, the 1H-1H COSY correlations (Figure S9) between H-1 and H-2，H-2 and H-3，H-3
and H-4，H-6 and H2–7 (1H, m, H-7α) /1.45 (1H, m, H-7β) were also observed. According to
these data, C-1 to C-6 was hydroxylated. The relative configuration of 1 based on the NOESY
correlations (Figure S10) of H-9α/H-1/H-2/H-3/H-4/H-6, Me-19β/H-8β and chiral carbon NMR
data. Thus, 1 was determined as lβ,2β,3β,4β,5β,6β–hexolhydroxy -pregn-16-en-20-one.
Additionally, the known steroidal saponins were identified by comparing their spectra
(NMR and MS) and physicochemical data with those reported in the literature as
ophiopogonin T (2) (Lee et al. 2016), (25S)-5β-spirostan-1β,2β,3β,4β,5β-pentol-5-O-β-D-
glucopyranoside (3) (Han et al. 2013), kitigenin5-O-β-D-glucopyranoside (4) (Wang, Hou

NATURAL PRODUCT RESEARCH
3
et al. 2013), (20S, 22R)-spirost-25(27)-en-1β,2β,3β,4β,5β-pentaol-5-O-β -D-glucopyranoside
(5) (Song, Li et al. 2015), (20S, 22R)-spirost-25(27)-en-1β, 3β,4β,5 β-tetraol-5-O-β-D-glucopyra-
noside (6) (Song, Li et al. 2015), (17,20-S-trans)-5β-pregn- 16-en-1β,3β-diol-20-one1-O-β-D-
xylopyranosyl-(2→1)-[α-L-rhamnopyranosyl]-3-O-α-L-rhamnopyranoside (7) (Zhang et al.
2016), (1β,3β,16β,22S)-cholest-5-ene-1,3, 16,22-tetrol 1,16-di(β-D-glucopyranoside) (8)
(Zhang et al. 2007), (1β,3β,16β,22S) -cholest-5-ene-1,3,16,22-tetrol 1-[O-α-L-rhamnopyranosyl-
(1→2)-β-D- glucopyrano side]16-(β-D-glucopyranoside) (9) (Zhang et al. 2007), dioscin (10)
(Shu et al. 2017) (Figure 1).
Compounds 1–10 were evaluated for anticomplement activity in vitro. The results were
summarised in Table S2. The compound 1 exhibited anticomplement effects with the CH50
values of 0.043 mg/mL. However, compounds 2–10 which are saponins showed no activity,
which was consistent with previous reports (Wen et al. 2017). Interestingly, hydrolysis of
steroidal saponins (2–10) resulted in its aglycones (2a–10a) (Figure S1) correspondingly,
identified by HR-ESI-MS data (Table S2), which exhibited anticomplement effects with CH50
values ranging from 0.049 to 0.156 mg/mL. Furthermore, the anticomplement activity was
positively correlated with the numbers of hydroxyl in steroids on the basis of comparing
compound 3 with 4, 5 with 6.
According to the relevant literatures (Wang, Huang et al. 2013; Han et al. 2010) , oral
saponins of R. carnea have good effect on anti-rheumatoid arthritis, anti-inflammatory and
pneumonia, while excessive complement activation may result in various inflammatory dis-
eases, including systemic lupus erythematosus (Morgan et al. 2005), rheumatoid arthritis
(Barilla-LaBarca et al. 2013), acute lung injury (Ballanti et al. 2011) and pneumonia. Therefore,
Figure 1. structures of compounds 1–10.

4
X. XU ET AL.
we deduced that saponins of R. carnea exerting pharmacological effect which may via
anticomplement. However, steroid saponins as the most abundant constituent in R. carnea
exhibited no activity in vitro. Moreover, most herbal medicines are orally administered, and
their components are brought into contact with intestinal microflora in the alimentary tract,
followed by intestinal bacteria transformation before exerting biological activities (Kim
et al. 1998; Xing et al. 2014). Therefore, we deduced that saponins of R. carnea may exert
pharmacological effect via anticomplement, and experiment of steroid saponins incubated
with intestinal bacteria will requires further investigation and confirmation.
3. Experimental
3.1. General experimental procedures
The IR spectra were recorded on a Bruker TENSOR-27 instrument. The HR-ESI-MS was meas-
ured on a Triple TOF™ 5600+ system with a Duo Spray source (AB SCIEX, Foster City, CA, USA).
1D and 2D NMR spectra were recorded on a Bruker-AVANCE600 instrument with TMS as an
internal standard. Optical rotations were recorded on a PerkinElmer Model 343 polarimeter.
The analytical HPLC was performed on a Shimadzu HPLC system (Kyoto, Japan) consisting
of a LC-20AD solvent delivery system, a SIL-20AC autosampler, a CTO-30A column oven, a
DGU-20A3 degasser and a CBM-20A controller, separation was performed on a Cosmosil C18
column (4.6 × 250 mm, 5.0 μm). Semipreparative HPLC was performed on a system compris-
ing a Waters 550 pump equipped with a Waters 2487 detector and a Cosmosil C18 column
(10 × 250 mm, 5.0 μm). Sephadex LH-20 gel was purchased from GE Health care Bio-Sciences
AB (Uppsala, Sweden). Macroporous adsorption resin HP20 as purchased from Mitsubishi
Chemical Corporation (Tokyo, Japan). Silica gel was purchased from Qingdao Haiyang
Chemical Group Corporation (Qingdao, China).
3.2. Plant material
Reineckia carnea (Andr.) Kunth. is a monotypic plant species in the Reineckia genus of the
Liliaceae family, which was identified by Vice Director of Pharmacists Bei Wu, Nanchang
Institute for Food and Drug Control. The material was collected from An’shun city in Guizhou
province, in March 2014. A voucher specimen (No. Z-140310-01) has been deposited at Jia
ngxi University of Traditional Chinese Medicine, Nan chang, China.
3.3. Animals and reagents for the anticomplement assay
Guinea pigs were purchased from Beijing Vital River Laboratory Animal Technology
Corporation Ltd. (Beijing, China). Sheep red blood cell (SRBC), hemolysin, and barbital buffer
solution (BBS) were purchased from Shanghai yuanye Biotechnology Corporation Ltd.
(Shanghai, China). Heparin sodium salt (≥140 IU/mg, dry basis) is a polyanionic glycosamino-
glycan from porcine intestinal mucosa and was purchased from Solarbio (Beijing, China).

NATURAL PRODUCT RESEARCH
5
3.4. Extraction and isolation
The air-dried whole plant of R. carnea (20 kg) was powdered and extracted with 80% EtOH
under reflux for three times at 80 °C. After removing the solvent, the concentrated residue
was successively partitioned with petroleum ether, EtOAc and n-BuOH . The n-BuOH-soluble
extract (812 g) was subjected to HP20 macroporous resin column eluted successively with
20–95% EtOH to give six fractions (Fr.1–Fr.6). Fr. 3 (78 g) was subjected to column chroma-
tography on ODS gel and further separated by repeated preparative HPLC to give compound
1 (10.2 mg; tR = 37 min). and compound 5 (5.8 mg; tR = 32 min), compound 6 (34.4 mg;
tR = 45 min), compound 7 (11.0 mg; tR = 28 min), compound 10 (5.8 mg; tR = 25 min). Fr. 4
(62 g) was also subjected to column chromatography on ODS gel and was purified by pre-
parative HPLC to obtain compound 2 (10.2 mg; tR = 18 min), compound 3 (4.8 mg; tR = 46 min),
compound 4 (4.5 mg; tR = 50 min), compound 8 (230.2 mg; tR = 40 min) and compound 9
(28.0 mg; tR = 26 min).
3.5. lβ,2β,3β,4β5β,6β-hexolhydroxy-pregn -16-en-20-one
A white amorphous powder; [ꢀ]2D0− 26.5° (c = 0.15, MeOH); IR (KBr) vmax: 3501, 3413, 2918,
1688, 1459, 1112 cm−1(Figure S3), 1H NMR (600 MHz, in CD3OD) and 13C NMR (150 MHz, in
CD3OD) spectral data, see Table S1; m/z 395.2067 [M − H]−(calcd for C21H31O7 395.2148).
3.6. Acid hydrolysis of compounds (2–10)
Compound 2–10 (4.0 mg, respectively) were hydrolysed with 1.5 mol/L HCl (CH3OH:H2O = 9:1)
and heated to 70 °C in a water bath for 2 h, respectively. The hydrolysed saponnins were
tested by TLC and HR-ESI-MS data (Table S2), and the corresponding aglycones (Figure S2)
were obtained from ethyl acetate extracting and evaluated with anticomplement activity.
3.7. Anticomplement assay through the classical pathway
On the basis of methods described previously by Song et al. (2014), sensitised erythrocytes
(EA) were prepared by incubation of sheep erythrocytes (4.0 × 108 cells/mL) with rabbit
anti-sheep erythrocyte antibodies in GVB2+, each compound and heparin (the positive con-
trol) were dissolved in GVB2+. Guinea pig serum (1:32), according to estimation of serum
potency, was chosen to give submaximal lysis in the absence of complement inhibitors. In
brief, various dilutions of test samples (200 μL) were preincubated with 200 μL of guinea pig
serum at 37 °C for 10 min. then, EA (200 μL) was added, and the mixture was incubated at
37 °C for 30 min. Controls [blank (200 μL of EA in 400 μL of GVB2+), 100% lysis (200 μL of EA
in 400 μL of H2O), and sample control (200 μL dilution of each sample in 400 μL of GVB2+)]
were incubated under the same conditions. The mixture was centrifuged (4 °C, 4000 rpm),
and the optical density of the supernatant (200 μL) was measured at 405 nm with a spec-
trophotometer (Thermo, Multiscan, MK3). Anticomplement activity was determined as the
mean of triplicate measurements at each concentration and expressed as 50% inhibitory
concentration (CH50 value).

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X. XU ET AL.
4. Conclusion
In conclusion, A new polyhydroxylated pregnane, named lβ,2β,3β,4β,5β,6β-hexolhydr oxy-
pregn-16-en-20-one, along with nine known steroidal saponins were isolated from the whole
plant of R. carnea. Compound 1 exhibited anticomplement effects with the CH50 values of
0.043 mg/mL, and saponins(2–10) showed no inhibition. However, hydrolysis of steroidal
saponins (2–10) resulted in its aglycones (2a–10a) correspondingly which exhibited anti-
complement activity with the CH50 values of 0.049–0.156 mg/mL.
Supplementary material
This consists of the abstract, TableS1–S2 and Figures S1–S10.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundations of China under [grant num-
ber 81560636], [grant number 81760702]; Jiangxi Province key R&D projects under [grant number
20165BCB19009]; Nanchang innovative talent team under [grant number 2016173].
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