Pentasaccharide resin glycosides with multidrug resistance reversal activities from the seeds of: Pharbitis nil

Article abstract of DOI:10.1039/c7ra09026a

Resin glycosides are novel P-glycoprotein inhibitors. In order to evaluate their multidrug resistance (MDR) reversal activities, we isolated seven new resin glycosides, pharbitins A-G (1-7) from the seeds of Pharbitis nil. Their chemical structures were determined by extensive application of high resolution 2D NMR techniques, HRESIMS and chemical methods. Compounds 1-4 and 6 were evaluated for their MDR reversal activities in KB/VCR, A549/T and K562/ADR cells. Among them, compound 2 showed moderate MDR reversal activity in KB/VCR cells, and increased the cytotoxicity of vincristine by 2.2-fold when incorporated at 25 μM. A structure-activity relationship study revealed that substituting Rha′′ C-3 with a trans-cinnamoyl group improves the MDR reversal activity. Also, an intracellular Rh123 accumulation assay demonstrated that compound 2 could inhibit the function of P-gp.

Full text of DOI:10.1039/c7ra09026a

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PAPER
Pentasaccharide resin glycosides with multidrug
resistance reversal activities from the seeds of
Pharbitis nil†
Cite this: RSC Adv., 2017, 7, 52001
Jun Li,ab Wen-Qiong Wang,ab Shuai Tang,ab Wei-Bin Song,ab Min Huangab
ab
*
and Li-Jiang Xuan
Resin glycosides are novel P-glycoprotein inhibitors. In order to evaluate their multidrug resistance (MDR)
reversal activities, we isolated seven new resin glycosides, pharbitins A–G (1–7) from the seeds of Pharbitis
nil. Their chemical structures were determined by extensive application of high resolution 2D NMR
techniques, HRESIMS and chemical methods. Compounds 1–4 and 6 were evaluated for their MDR
reversal activities in KB/VCR, A549/T and K562/ADR cells. Among them, compound 2 showed moderate
MDR reversal activity in KB/VCR cells, and increased the cytotoxicity of vincristine by 2.2-fold when
incorporated at 25 mM. A structure–activity relationship study revealed that substituting Rha00 C-3 with
a trans-cinnamoyl group improves the MDR reversal activity. Also, an intracellular Rh123 accumulation
assay demonstrated that compound 2 could inhibit the function of P-gp.
Received 15th August 2017
Accepted 26th October 2017
DOI: 10.1039/c7ra09026a
rsc.li/rsc-advances
seven new resin glycosides pharbitins A–G (1–7) (Fig. 1) with
macrolactone rings from the seeds of P. nil. According to their
Introduction
structures, the new compounds can be divided into two types:
those possessing 18-membered rings (1–5), and compounds 6–7
with 19-membered rings. Among them, 2 showed moderate
activity in MDR reversal against KB/VCR cells, and increased the
cytotoxicity of vincristine by 2.2-fold when incorporated at 25
mM. Herein, we described the isolation, structural elucidation
and MDR reversal activity evaluation of all isolates from the
seeds of P. nil.
Resin glycosides are unusual amphipathic metabolites with
structures including hydrophobic (fatty acid aglycone) and
hydrophilic (oligosaccharide) moieties, which are mainly found
in the family Convolvulaceae.1 Resin glycosides exhibit various
pharmacological activities, such as cytotoxicity, multidrug
resistance (MDR) reversal, ionophoresis, and phytogrowth–
inhibitory activities.2–4 Because of their intriguing structures
and varied activities, resin glycosides have attracted more
attention in recent times. Moreover, as novel P-glycoprotein
inhibitors, the resin glycosides from Merremia hederacea with
MDR reversal activities have been investigated in our previous
work.5 However, considering the unclear structure–activity
relationship (SAR) and pharmacological mechanism of the
resin glycosides, we intend to isolate various analogues from the
family Convolvulaceae, investigate their MDR reversal activities,
and then proceed to establish their SAR and pharmacological
mechanism.
Results and discussion
Pharbitin A (1) was obtained as a colorless gum. The molecular
formula was established as C66H104O27 based on 13C NMR data
(Table 3) and HRESIMS ion at m/z 1351.6647 [M + Na]+ (calcd for
C
66H104O27Na, 1351.6657). Its IR spectrum exhibited absorp-
tions of hydroxyl (3426 cmꢀ1), carbonyl (1724 cmꢀ1), and
aromatic (1635 cmꢀ1) groups. The NMR spectra showed ve
anomeric signals [dH 4.90 (d, J ¼ 7.5 Hz), dH 5.12 (d, J ¼ 7.6 Hz),
dH 5.59 (d, J ¼ 1.8 Hz), dH 5.85 (d, J ¼ 1.8 Hz), dH 6.29 (d, J ¼ 1.8
Hz)] and dC 166.8, 173.7, 173.9, 176.3] and signals of long-chain
fatty acids, which indicated that 1 was a resin glycoside.9 The
NMR data of 1 could be divided into two parts: resonances in
the anomeric region and those representative of the aglycone
moieties.
Pharbitidis Semen, the seeds of Pharbitis nil, is a purgative
crude drug widely grown in China and Japan.6 So far, only four
pure resin glycoside acids (pharbitic acids A–D) with an acyclic
core have been reported from P. nil.6–8 We report the isolation of
aState Key Laboratory of Drug Research, Shanghai Institute of Meteria Medica, Chinese
Academy of Sciences, 501 Haike Road, Shanghai 201201, People's Republic of China.
E-mail: ljxuan@simm.ac.cn; Fax: +86-21-20231968; Tel: +86-21-20231968
bUniversity of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049,
People's Republic of China
For the aglycone part, the 1H NMR data of 1 (Table 1)
exhibited two trans-coupled olenic protons at dH 6.59 (d, J ¼
16.0 Hz) and dH 7.85 (d, J ¼ 16.0 Hz) due to trans-cinnanoyl
group (CA). The signals at dH 0.79 (t, J ¼ 7.0 Hz) and dH 2.31 (m)
due to the n-octanoyl group (Octa). Also a methyl triplet signal at
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra09026a
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Fig. 1 Chemical structures of compounds 1–7.
dH 0.82, a methyl doublet signal at dH 1.14 and a methine by the correlation between C-1 of Con (dC 173.7) and H-2 of a-Rha
multiplet signal at dH 2.49 due to 2-methybutanoyl moiety (2- (dH 6.02). Thus, the structure of compound 1 was identied as
Mba). Aer alkaline hydrolysis, the S absolute conguration of (11S)-convolvulinolic acid 11-O-b-D-glucopyranosyl-(1 / 3)-O-[3-
2-Mba was determined by chiral gas chromatography (GC) O-(trans-cinnamoyl)-4-O-(2S-methylbutanoyl)-a-L-rhamnopyr-
analysis. In addition, the 11-hydroxytetradecanoic acid moiety anosyl-(1 / 4)]-O-[2-O-n-octanoyl]-a-L-rhamnopyranosyl-(1 /
(convolvulinolic acid, Con) was suggested by the diagnostic 4)-O-a-L-rhamnopyranosyl-(1 / 2)-O-b-D-glucopyranoside-(1, 20-
signals of methyl triplet at dH 0.85, methylene group at dH 2.46 lactone).
and 2.34, and oxygenated methine at dH 3.88. Aer alkaline and
The molecular formulas of pharbitins B–E (2–5) were deter-
acid hydrolysis, the resulting 11-hydroxytetradecanoic acid mined as C68H108O27, C57H98O26, C59H102O26 and C64H112O26,
showed a fragment ion at m/z 183 [M ꢀ CH3(CH2)2 ꢀ H2O]+ by respectively, based on the 13C NMR (Table 3) and HRESIMS
the EIMS data, suggesting the 11-OH group of Con. Its absolute data. The 1H and 13C NMR spectra indicated that 2–5 consisted
conguration was determined to be S by the Mosher's method.10 of the same complex sugar moiety and (11S)-hydroxyte-
In the anomeric region, the 1H–1H COSY data (Fig. 2) indicated tradecanoyl group as 1. They differed at the kinds of acyl residue
ve spin systems, which were attributed to three 6-deoxyhexose and the sites of acylation. The identities of the acyl moieties
and two hexose units. The sugars obtained from the acidic were determined by LC-HRMS aer basic hydrolysis. 2-Mba, CA,
hydrolysates were identied as L-rhamnose and D-glucose through Deca (n-decanoic acid) in 2, 2-Mba, Octa in 3, 2-Mba, Deca in 4
the HPLC analysis and their corresponding optical rotations. The and Deca in 5 were identied. The positions of acylation were
b-congurations of D-glucose was determined by a large coupling determined by the HMBC correlations as follows: OH-4 of Rha00
constant (J ¼ 7.5 Hz and 7.6 Hz) for the anomeric protons at dH was acylated by 2-Mba in 2–4 and by Deca in 5, OH-2 of Rha0 was
4.90 and dH 5.12 in the 1H NMR spectrum, while a-conguration acylated by Deca in 2, 4, 5 and by Octa in 3, OH-3 of Rha00 was
of L-rhamnose was revealed by the chemical shi of C-5 of acylated by CA only in 2. Moreover, the site of lactonization was
13
rhamnose (dC 69.3, 68.8, 68.5) in the C NMR spectrum.11 The corroborated as C-2 of Rha according to the correlation between
long-range HMBC correlations (Fig. 2) between H-1 of b-Glc (dH C-1 of Con and H-2 of Rha. Consequently, the structures of 2–5
4.90) and C-11 (dC 82.7) of the 11-hydroxytetradecanoyl moiety were determined as shown.
indicated that b-Glc was the rst hexose unit in the sugar moiety.
Pharbitin F (6) has the same molecular formula, C66H104O27,
The sequence of the sugar moiety was determined to be glucosyl- as 1, according to its HRESIMS data at m/z 1351.6663 [M + Na]+
(1 / 3)-[rhamnosyl-(1 / 4)]-rhamnosyl-(1 / 4)-rhamnosyl-(1 / (calcd 1351.6657). Alkaline hydrolysis of 1 and 6 afforded 2-
2)-glucosyl by their long-range HMBC correlations: H-1 of a-Rha Mba, CA and Octa in the CHCl3 layer. The sites of acylation of 6
(dH 5.59) to C-2 of b-Glc (dC 82.2), H-1 of a-Rha0 (dH 5.85) to C-4 of were the same as 1, which was supported by the key HMBC
a-Rha (dC 82.2), H-1 of a-Rha00 (dH 6.29) to C-4 of a-Rha0 (dC 79.3), correlation from Rha00 H-4 (dH 6.06) to 2-Mba C-1 (dC 176.3),
and dH H-1 of b-Glc0 (dH 5.12) to C-3 of a-Rha0 (dC 80.2). In addi- Rha00 H-3 (dH 5.93) to CA C-1 (dC 166.5), Rha0 H-2 (dH 6.03) to
tion, the positions of esterication, i.e., 2-Mba located at OH-4 of Octa C-1 (dC 173.9). Moreover, 1H and 13C NMR data of 1 and 6
a-Rha00, CA at OH-3 of a-Rha00, Octa at OH-2 of a-Rha0 were indicated that they share the same pentasaccharide skeleton.
inferred from the long-range correlations: H-4 of a-Rha00 (dH 6.09) The only difference between 1 and 6 was the position of the
to C-1 of 2-Mba (dC 176.3), and H-3 of a-Rha00 (dH 5.99) to C-1 of CA lactonization. In the HMBC spectrum of 6, the H-3 proton of
(dC 166.8), H-2 of a-Rha0 (dH 6.34) to C-1 of Octa (dC 173.9) Rha resonated at dH 5.70 and showed an HMBC correlation to
respectively. The C-2 (Rha) site of lactonization was corroborated the carbonyl group that resonated at dC 175.0 (C-1 of Con),
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Table 1 1H NMR (500 MHz) data of 1–5 (in ppm) (pyridine-d5)a
Position
1
2
3
4
5
Glc-1
4.90 d (7.5)
3.89 m*, b
4.18 m*
4.12 m*
3.85 m*
4.91 d (7.4)
3.90 m*
4.19 m*
4.13 m*
3.86 m*
4.88 d (7.5)
3.87 m*
4.14 m*
4.11 m*
3.83 m*
4.86 d (7.5)
3.86 m*
4.14 m*
4.10 m*
3.82 m*
4.89 d (7.4)
3.89 m*
4.17 m*
4.13 m*
3.84 m*
2
3
4
5
6
4.45 m*, 4.30 dd
(11.7, 5.2)
4.46 m*, 4.31 dd
(12.2, 5.2)
4.45 m*, 4.30 dd
(12.5, 4.0)
4.44m*, 4.28 dd
(11.9, 5.5)
4.46m*, 4.32 dd
(10.7, 5.1)
Rha-1
2
3
4
5.59 d (1.8)
6.02 dd (3.4, 1.8)
5.07 m*
5.60 d (1.8)
6.03 dd (3.4, 1.8)
5.07 m*
5.58 d (1.8)
6.02 dd (3.3, 1.8)
5.04 m*
5.56 d (1.8)
6.01 dd (3.3, 1.8)
5.03 m*
5.59 d (1.8)
6.04 dd (3.4, 1.8)
5.03–5.09 m
4.19 m*
4.16 m*
4.17 m*
4.18 m*
4.17 m*
5
4.44 m*
4.45 m*
4.43 m*
4.42 m*
4.44 m*
6
1.60 d (6.2)
5.85 d (1.8)
6.34 br s
4.81 dd (8.9, 3.3)
4.37 m*
1.61 d (6.1)
5.86 d (1.9)
6.35 dd (3.2, 1.9)
4.80–4.86 m
4.38 m*
1.60 d (6.2)
5.91 d (1.9)
6.30 dd (3.4, 1.9)
4.75–4.81 m
4.34 m*
1.59 d (6.1)
5.91 d (1.8)
6.29 br s
4.74–4.80 m
4.33 m*
1.62 d (6.0)
5.94 d (1.9)
6.33 dd (3.4, 1.9)
4.77–4.83 m
4.36 m*
Rha0-1
2
3
4
5
4.36 m*
4.37 m*
4.35 m*
4.34 m*
4.37 m*
6
1.65 d (5.7)
6.29 d (1.8)
5.27 dd (3.1, 1.8)
5.99 dd (9.8, 3.1)
6.09 t (9.8)
4.50 dd (9.8, 6.3)
1.43 d (6.3)
5.12 d (7.6)
3.96 m*
1.66 d (5.4)
6.30 d (1.8)
5.28 br s
6.00 dd (9.8, 3.1)
6.10 t (9.8)
4.51 dd (9.8, 6.3)
1.44 d (6.3)
5.13 d (7.6)
3.97 m*
1.66 d (5.1)
6.24 d (1.9)
4.95 br s
4.49–4.57 m
5.76 t (9.4)
4.36 m*
1.40 d (6.2)
5.08 d (7.7)
3.96 m*
1.64 d (5.5)
6.23 d (1.9)
4.94 br s
4.51 dd (9.4, 3.8)
5.75 t (9.4)
4.35 m*
1.39 d (6.2)
5.07 d (7.6)
3.95 m*
1.67 d (5.5)
6.28 d (1.9)
4.99 m*
4.55–4.61 m
5.82 t (9.4)
4.38 m*
1.45 d (6.3)
5.11 d (7.6)
3.98 m*
Rha00-1
2
3
4
5
6
Glc0-1
2
3
4.06 m*
4.07 m*
4.03 m*
4.02 m*
4.05 m*
4
3.93 m*
3.94 m*
3.94 m*
3.93 m*
3.96 m*
5
6
3.76–3.82 m
4.40 m*, 4.09 m*
2.46 m*, 2.34 m*
3.88 m*
3.80 ddd (9.8, 5.9, 2.5)
4.41 m*, 4.10 m*
2.47 m*, 2.36 m*
3.89 m*
3.74 ddd (8.9, 5.8, 2.5)
4.38 m*, 4.08 m*
2.46 m*, 2.35 m*
3.86 m*
3.73 ddd (8.9, 5.8, 2.5)
4.37 m*, 4.07 m*
2.45 m*, 2.34 m*
3.85 m*
3.76 ddd (8.9, 5.9, 2.5)
4.41 m*, 4.10 m*
2.45 m*, 2.37 m*
3.88 m*
Con-2
11
14
2-Mba-2
3
0.85 t (6.9)
2.49 m*
0.85 t (7.3)
2.50 m*
0.85 t (7.3)
2.51 m*
1.78 m*, 1.49 m*
0.93 t (7.4)
1.20 d (7.0)
0.83 t (7.2)
2.50 m*
1.77 m*, 1.47 m*
0.92 t (7.4)
1.19 d (7.0)
0.85 t (7.1)
1.70 m*, 1.40 m*
0.82 t (6.9)
1.14 d (6.9)
6.59 d (16.0)
7.85 d (16.0)
7.45 2H m
7.34 2H m*
7.34 m*
1.71 m*, 1.41 m*
0.82 t (7.5)
1.15 d (7.0)
6.59 d (16.0)
7.86 d (16.0)
7.46 2H m
7.34 2H m*
7.34 m*
4
2-Me
CA-2
3
20/60
30/50
40
Octa-2
8
2.31 m*
0.79 t (7.0)
2.31 m*
0.79 t (6.9)
Deca-2
10
2.33 m*
0.85 t (7.2)
2.30 m*
0.83 t (7.2)
2.32 m*
0.85 t (7.1)
2.48 m*
Deca0-2
10
0.85 t (7.1)
a
Chemical shis (ppm) referenced to pyridine-d5 (dH 7.58) at 500 MHz. b Chemical shis marked with an asterisk (*) indicate overlapped signals.
which suggested the lactone bond was linked at C-3 of Rha in 6 (2-Mba, Deca) and same sites of acylation (OH-4 of Rha00 was
rather than at C-2 of Rha as in 1. Therefore, the structure of 6 acylated by 2-Mba, OH-2 of Rha0 was acylated by Deca). The only
was determined as shown.
difference between 4 and 7 was the position of the lactonization.
Pharbitin G (7) has the same molecular formula, C59H102O26, The lactonization site was bonded at C-3 of Rha for 7, while at C-
as 4, according to its HRESIMS data at m/z 1249.6550 [M + Na]+ 2 of Rha for 4, on the basis of corresponding HMBC correlation:
(calcd 1249.6552). 1D NMR and the HMBC spectrum, together dH 5.68 (Rha H-3) to dC 175.0 (Con C-1) in 7, dH 6.01 (Rha H-2) to
with the alkaline hydrolysis analysis, suggested 4 and 7 share dC 173.7 (Con C-1) in 4. Thus, the structure of 7 was determined
the same pentasaccharide skeleton, same acyl functions as shown.
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Table 2 1H NMR (500 MHz) data of 6–7 (in ppm) (pyridine-d5)a
The cytotoxicity assay showed that the inhibition ratios of
2 and 4 were less than 50% at 25 mM, indicating that these
compounds were noncytotoxic at 25 mM. However, the inhi-
bition ratios of 1, 3, 6 were more than 50% at 25 mM, indi-
cating that these compounds were cytotoxic at 25 mM, thus,
these compounds were tested at 5 mM. Compounds 2 and 4
enhanced the cytotoxicity of vincristine by 1.1–2.2-fold when
incorporated at 25 mM, while compound 1, 3, 6 increased
the cytotoxicity of vincristine by 0.9–1.3-fold when incorpo-
rated at 5 mM. Compound 2 with a CA substituent group at
Rha00 C-3 was 2 times more active than compound 4 with no
substituent group at the same site. Similar result was
observed for compound 1 and 3, which demonstrated that,
substituting Rha00 C-3 with a CA group improves MDR reversal
activity.
Moreover, compound 2 was tested for its effects on both P-
glycoprotein (P-gp) function and expression (Fig. 3 and 4).
Intracellular rhodamine 123 (Rh123)-associated mean uores-
cence intensity in KB and KB/VCR cells was used to study the
effects of compound on the inhibition of P-gp function. The
result showed that the Rh123 accumulation of KB/VCR was a lot
less than the parental KB cells and the Rh123 accumulation of
KB/VCR pretreated with compound 2 was twice higher than
untreated. However, the expression of P-gp detected by Western
blot showed that P-gp was overexpressed in KB/VCR cell and
compound 2 had no effect on P-gp expression.
Position
6
7
Glc-1
2
3
5.04 d (7.8)
4.28 m*, b
4.37 m*
5.03 d (8.0)
4.28 m*, b
4.35 m*
4
4.18 m*
4.17 m*
5
3.91 m*
3.91 m*
6
4.56 m*, 4.38 m*
6.47 d (1.8)
5.24 br s
4.50 m*, 4.38 m*
6.48 d (1.7)
5.25 br s
Rha-1
2
3
4
5
5.70 dd (10.0, 2.8)
4.71 m*
5.07 m*
5.68 dd (9.7, 2.7)
4.72 t (9.7)
5.06 m*
6
1.72 d (6.2)
5.63 d (1.8)
6.03 dd (3.4, 1.8)
4.74 dd (8.9, 3.4)
4.37 m*
1.70 d (6.1)
5.65 d (1.8)
6.01 dd (3.5, 1.8)
4.66 dd (8.8, 3.5)
4.36 m*
Rha0-1
2
3
4
5
4.44 m*
4.39 m*
6
1.66 d (5.8)
6.28 d (2.5)
5.22 br s
1.65 d (5.9)
6.22 d (2.0)
4.93 br s
Rha00-1
2
3
4
5
5.93 dd (9.8, 2.5)
6.06 t (9.8)
4.47 m*
4.47 m*
5.75 t (9.7)
4.36 m*
6
1.44 d (6.2)
5.15 d (7.7)
3.96 m*
1.41 d (6.2)
5.10 d (7.6)
3.97 m*
Glc0-1
2
3
4.16 m*
4.14 m*
4
4.18 m*
4.17 m*
5
3.91 m*
3.91 m*
Conclusions
6
4.56 m*, 4.38 m*
2.74 ddd
4.50 m*, 4.38 m*
2.70 ddd
Con-2
The chemical investigation of the seeds of P. nil led to the
isolation of seven new pentasaccharide resin glycosides
pharbitins A–G (1–7), and it is the rst time resin glycosides
with macrolactone rings is being reported from P. nil. The
isolated resin glycosides shared the same oligosaccharide
core, and differed at the kinds of acyl residue, and the sites of
acylation. According to the sites of acylation, compounds 1–5
possess 18-membered rings, whereas compounds 6–7 possess
19-membered rings. Compound 2 showed moderate MDR
reversal activity in KB/VCR cell, and increased the cytotoxicity
of vincristine by 2.2-fold when incorporated at 25 mM. SAR
study showed that, substituting Rha00 C-3 with a CA group
improves MDR reversal activity. This work therefore
contributes to the SAR study of resin glycosides with MDR
reversal activities. Furthermore, we found that compound 2
inhibited the function of P-gp but had no effect on P-gp
expression.
(14.2, 10.8, 2.7), 2.32 m*
3.94 m*
1.02 t (7.0)
(14.2, 10.7, 2.8), 2.28 m*
3.93 m*
0.95 t (7.1)
11
14
Jal-2
11
14
2-Mba-2
3
4
2.46 m*
2.50 m*
1.77 m*, 1.48 m*
0.93 t (7.4)
1.69 m*, 1.40 m*
0.81 t (7.4)
1.13 d (7.0)
6.56 d (16.0)
7.85 d (16.0)
7.44 2H m
7.33 2H m*
7.33 m*
2-Me
CA-2
3
1.19 d (7.1)
20/60
30/50
40
Octa-2
8
Deca-2
10
2.42 m*
0.81 t (6.9)
2.43 m*
0.85 t (7.1)
a
Chemical shis (ppm) referenced to pyridine-d5 (dH 7.58) at 500 MHz.
Chemical shis marked with an asterisk (*) indicate overlapped
b
signals.
Experimental
General experimental procedures
Isolates (1–4 and 6) were examined for their multidrug
resistance (MDR) reversal activities in KB/VCR and A549/T cells
using the SRB method, while in K562/ADR cell using the MTT
Optical rotations were determined with a Perkin Elmer 341
polarimeter. UV spectra were obtained on a Shimadzu UV-2450
spectrophotometer. IR spectra were measured by a Bruker
method (Tables 4 and S1†). However, only the KB/VCR cell line Tensor 27 spectrometer. NMR experiments were recorded in
showed favorable results and as such we proceeded to carry out pyridine-d5 on Bruker AM-400 and Bruker AM-500 spectrome-
further experiments with this cell line. The MDR reversal ters referenced to solvent peaks (dH 7.58; dC 135.9). EIMS anal-
activity in KB/VCR is described below.
yses were performed on a Thermo-DFS mass spectrometer.
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Fig. 2 Key HMBC and 1H–1H COSY correlations of pharbitins A–G (1–7).
HRESIMS analyses were performed on an Agilent 6224 TOF
mass spectrometer with an ESI interface. Semipreparative HPLC
was carried out on an Agilent 1100 series HPLC system with
a YMC-Pack ODS-A column (250 ꢁ 10 mm, 5 mm). Analytical
HPLC was performed on an Agilent 1200 series HPLC system
with an Agilent ZORBAX NH2 column (150 ꢁ 4.6 mm, 5 mm).
Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd.,
People's Republic of China), C18 reversed-phase (RP-18) silica
gel (20–45 mm; Fuji Silysia Chemical Ltd., Japan) and Sephadex
LH-20 gel (Amersham Biosciences, Sweden) were used for
column chromatography (CC). Pre-coated silica gel GF254
plates (Qingdao Haiyang Chemical Co. Ltd., People's Republic
of China) were used for TLC.
Plant material
Seeds of Pharbitis nil were collected in Guangxi, China, in
August 2013 and identied by Prof. Heming Yang. A voucher
specimen (no. SIMM810) was deposited at the Herbarium of the
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences and People's Republic of China.
Extraction and isolation
Air–dried, powdered seeds of Pharbitis nil (5.0 kg) were reuxed
with 95% EtOH three times and concentrated in vacuo to give
a crude extract (230 g), which was suspended in 2 L of water and
then extracted with EtOAc to give the EtOAc-soluble fraction
(168 g). The EtOAc layer was concentrated, and subjected to
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Table 3 13C NMR (125 MHz) data of 1–7 (in ppm) (pyridine-d5)a
Position
1
2
3
4
5
6
7
Glc-1
2
3
4
5
6
Rha-1
2
3
4
104.6
82.2
76.9
72.0
78.3
63.1
98.8
73.8
69.6
82.2
69.3
19.3
100.5
73.6
80.2
79.3
68.8
19.4
103.7
70.2
73.3
72.2
68.5
18.2
105.8
75.5
78.8
71.8
78.4
63.3
173.7
34.6
82.7
14.9
176.3
41.9
27.3
12.1
17.3
166.8
118.9
145.7
135.1
128.8
129.6
131.1
173.9
34.9
14.6
104.6
82.3
76.9
72.0
78.4
63.2
98.8
73.9
69.7
82.2
69.4
19.4
100.6
73.6
80.3
79.3
68.8
19.4
103.7
70.3
73.4
72.2
68.5
18.3
105.9
75.6
78.8
71.9
78.5
63.3
173.7
34.6
82.7
14.9
176.3
41.9
27.4
12.2
17.3
166.8
118.9
145.7
135.1
128.9
129.6
131.1
104.6
82.3
76.9
72.2
78.3
63.2
98.9
73.9
69.8
81.6
69.3
19.2
100.3
73.5
80.5
79.0
68.8
19.5
103.6
72.8
70.7
75.5
68.5
18.4
105.8
75.6
78.8
71.8
78.5
63.3
173.7
34.6
82.7
14.9
176.7
41.9
27.5
12.1
17.4
104.6
82.3
76.9
72.1
78.2
63.1
98.8
73.9
69.7
81.6
69.3
19.2
100.2
73.4
80.4
78.9
68.8
19.4
103.5
72.8
70.6
75.5
68.5
18.3
105.7
75.5
78.7
71.7
78.4
63.2
173.7
34.6
82.7
14.8
176.6
41.9
27.4
12.1
17.3
104.7
82.3
77.0
72.2
78.4
63.2
98.9
73.9
69.8
81.5
69.4
19.3
100.3
73.5
80.6
78.9
68.9
19.5
103.6
72.8
70.7
75.9
68.6
18.4
105.8
75.6
78.9
71.8
78.6
63.3
173.7
34.6
82.8
14.9
101.7
75.6
79.9
72.2
78.4
63.1
100.3
70.2
78.1
77.7
68.5
19.6
99.8
72.6
80.5
79.4
68.5
19.2
103.7
70.2
73.2
72.4
68.5
18.3
105.1
75.9
78.8
71.2
78.5
62.9
175.0
34.5
79.8
15.2
176.3
41.9
27.3
12.1
17.2
166.5
119.0
145.6
135.1
128.8
129.6
131.0
173.9
34.8
14.6
101.7
75.8
79.7
72.4
78.5
63.2
100.5
70.2
78.3
77.1
68.5
19.6
99.7
72.5
80.7
78.9
68.6
19.1
103.8
72.7
70.6
75.6
68.5
18.4
105.2
75.6
78.6
71.1
78.8
62.9
175.0
34.5
80.0
15.1
176.7
41.9
27.4
12.1
17.4
5
6
Rha0-1
2
3
4
5
6
Rha00-1
2
3
4
5
6
Glc0-1
2
3
4
5
6
Con-1
2
11
14
2-Mba-1
2
3
4
5
CA-1
2
3
10
20/60
30/50
40
Octa-1
2
8
174.0
34.9
14.6
Deca-1
2
173.9
34.9
14.6
174.0
34.9
14.6
174.0
34.9
14.7
173.9
35.1
14.7
174.0
34.9
14.7
10
Deca0-1
2
10
a
Chemical shis (ppm) referenced to pyridine-d5 (dC 135.9) at 125 MHz.
silica gel CC, eluting with petroleum ether/acetone in a gradient Fraction G3 was further subjected to a Sephadex LH-20 gel
manner (100 : 0 to 0 : 100, v/v) to afford nine fractions (A-I). column followed by a RP-C18 column using a gradient of
Fraction G was subjected to silica gel CC and eluted with MeOH/H2O (80 : 20 to 100 : 0, v/v) to give 6 (12 mg). Fraction H
CHCl3/MeOH (20 : 1 to 1 : 1) to yield subfractions (G1–G7). was applied to silica gel CC and eluted with CHCl3/MeOH (20 : 1
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Table 4 Results of modulating MDRa Activities in KB/VCR cells of
compounds 1–4 and 6
method was applied to fraction H4 to yield compound 5 (10 mg).
Fraction H5 was subjected to a RP-C18 column (50 : 50 to
100 : 0, v/v) to give ve subfractions (H5A-H5E). Fraction H5D
was chromatographed on a silica gel column and eluted with
CHCl3/MeOH (10 : 1 to 5 : 1) to give fraction H5D1 and H5D2,
which were further puried on an open ODS column (MeOH/
H2O, 85 : 15 to 100 : 0, v/v) to obtain 3 (60 mg) and 4 (60 mg),
respectively.
Vincristine + Sampleb
Inhibition ratio
Sample
% (25 mM)
IC50 value (mM)
RFc value
2
4
5.62
4.17
0.22
0.44
0.48
2.2
1.1
Vincristine
Pharbitin A (1). Colorless gum; [a]2D6 ꢀ29.5 (c 0.1, MeOH); UV
(MeOH) lmax (log 3) 203 (3.89), 217 (3.91), 280 (4.09) nm; IR
Vincristine + Sampled
Inhibition ratio
(KBr) vmax 3426, 2926, 2855, 1724, 1635, 1052 cmꢀ1; 1H and 13
C
Sample
% (5 mM)
IC50 value (mM)
RFe value
NMR data, see Tables 1 and 3; HRESIMS m/z 1351.6647 [M +
Na]+ (calcd for C66H104O27Na, 1351.6657).
1
3
6
2.84
1.85
10.64
0.36
0.53
0.37
0.48
1.3
0.9
1.3
Pharbitin B (2). Colorless gum; [a]2D6 ꢀ33.0 (c 0.1, MeOH); UV
(MeOH) lmax (log 3) 203 (3.66), 217 (3.64), 279 (3.80) nm; IR
Vincristine
(KBr) vmax 3429, 2929, 2858, 1730, 1632, 1049 cmꢀ1; 1H and 13
C
a
MDR: multidrug resistance. b Serial dilutions ranging from 0.125 to 1
mM of vincristine in the presence or absence of 25 mM sample. c RF: IC50
of VCR alone/IC50 of VCR in presence of 25 mM sample. d Serial dilutions
ranging from 0.125 to 1 mM of vincristine in the presence or absence of 5
NMR data, see Tables 1 and 3; HRESIMS m/z 1379.6988 [M +
Na]+ (calcd for C68H108O27Na, 1379.6970).
Pharbitin C (3). Colorless gum; [a]2D6 ꢀ23.7 (c 0.1, MeOH); UV
(MeOH) lmax (log 3) 203 (3.64), 275 (2.45) nm; IR (KBr) vmax 3405,
2929, 2852, 1727, 1046 cmꢀ1; 1H and 13C NMR data, see Tables 1
e
mM sample. RF: IC50 of VCR alone/IC50 of VCR in presence of 5 mM
sample.
and 3; HRESIMS m/z 1221.6258 [M
+
Na]+ (calcd for
C
57H98O26Na, 1221.6239).
Pharbitin D (4). Colorless gum; [a]2D7 ꢀ29.8 (c 0.1, MeOH); UV
(MeOH) lmax (log 3) 203 (3.59), 275 (2.43) nm; IR (KBr) vmax 3417,
2926, 2855, 1727, 1052 cmꢀ1; 1H and 13C NMR data, see Tables 1
and 3; HRESIMS m/z 1249.6561 [M
+
Na]+ (calcd for
C
59H102O26Na, 1249.6552).
Pharbitin E (5). Colorless gum; [a]2D7 ꢀ35.0 (c 0.1, MeOH); UV
(MeOH) lmax (log 3) 203 (3.43), 275 (2.38) nm; IR (KBr) vmax 3396,
2926, 2855, 1727, 1046 cmꢀ1; 1H and 13C NMR data, see Tables 1
and 3; HRESIMS m/z 1277.6862 [M
+
Na]+ (calcd for
C
61H106O26Na, 1277.6865).
Pharbitin F (6). Colorless gum; [a]2D5 ꢀ35.0 (c 0.1, MeOH); UV
Fig. 3 Effects of compound 2 on Rh123 accumulation in drug-
sensitive KB and multidrug-resistant KB/VCR cells. Cells were
respectively untreated (A) and pretreated with 25 mM of compound 2
(B).
(MeOH) lmax (log 3) 203 (3.83), 217 (3.85), 280 (4.05) nm; IR
(KBr) vmax 3417, 2926, 2861, 1730, 1635, 1043 cmꢀ1; 1H and 13
C
NMR data, see Tables 2 and 3; HRESIMS m/z 1351.6663 [M +
Na]+ (calcd for C66H104O27Na, 1351.6657).
Pharbitin G (7). Colorless gum; [a]2D6 ꢀ48.0 (c 0.1, MeOH); UV
(MeOH) lmax (log 3) 203 (3.57), 275 (2.66) nm; IR (KBr) vmax 3399,
2926, 2855, 1727, 1049 cmꢀ1; 1H and 13C NMR data, see Tables 2
and 3; HRESIMS m/z 1249.6550 [M
59H102O26Na, 1249.6552).
+
Na]+ (calcd for
C
Aglycon identication
Fig. 4 Effects of compound 2 on MDR-1 protein expression in drug-
Compounds 1–7 (5 mg, each) were separately dissolved in 5%
sensitive KB and multidrug-resistant KB/VCR cells. Cells were treated
with compound 2 and MDR-1 protein expression was analyzed by NaOH (4 mL) and reuxed at 90 ꢂC for 4 h. Each of the reaction
Western blot.
mixtures were acidied to PH 4, followed by extraction with
CHCl3 (3 ꢁ 4 mL) and concentration.12 The CHCl3 layer was
then analyzed by HRESIMS to identify organic acids: 2-methyl-
butanoic acid (m/z 101.0606 [M ꢀ H]ꢀ, calcd 101.0608) in 1–4
to 1 : 1) to afford subfractions (H1–H6). Fraction H2 was further
and 6–7; n-octanoic acid (m/z 143.1080 [M ꢀ H]ꢀ, calcd
puried by a Sephadex LH-20 gel column followed by prepara-
143.1078) in 1, 3 and 6; n-decanoic acid (m/z 171.1390 [M ꢀ H]ꢀ,
tive HPLC (MeOH/H2O, 100 : 0, v/v) to obtain 1 (60 mg) and 2
(60 mg). Fraction H3 was chromatographed on a Sephadex
LH-20 gel column followed by a RP-C18 column using a gradient
of MeOH/H2O (80 : 20 to 100 : 0, v/v) to yield 7 (8 mg). Same
calcd 171.1391) in 2, 4, 5 and 7; trans-cinnamic acid (m/z
147.0453 [M ꢀ H]ꢀ, calcd 147.0452) in 1, 2 and 6. The CHCl3
layer was also analyzed by chiral GC to determined the absolute
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conguration of 2-methylbutanoic acid. In addition, the were measured using a MTT assay. Briey, cells were plated in
aqueous phase via CHCl3 extraction was then extracted with 96-well culture plates for 24 h and treated with serial dilutions
n-BuOH (3 ꢁ 4 mL) and concentrated to yield a colorless solid. of adriamycin (Selleck) ranging from 3.1 to 100 mM. Then cells
The residue in 1 N H2SO4 (4 mL) was reuxed at 90 C for 4 h. were added 10 mL 0.5 mg mLꢀ1 MTT and incubated for 4 h. At
ꢂ
The reaction mixture was extracted with CHCl3 (3 ꢁ 4 mL), last, DMSO was added followed by detection of the absorbance
which was further concentrated and puried to afford 11- value at a wavelength of 570 nm.
hydroxytetradecanoic acid in 1–7. The 11-hydroxytetradecanoic
The results were expressed as IC50 values. The reversal fold
acid was further separately treated with (S)-MPTA chloride and (RF) as potency of reversal was obtained from tting the
(R)-MPTA chloride in pyridine-d5, and allowed to stand for 12 h data to RF ¼ IC50 of vincristine or taxel or adriamycin alone/
at room temperature. The S conguration of 11-hydroxyte- IC50 of vincristine or taxel or adriamycin in the presence of
tradecanoic acid was determined by the chemical shi differ- sample.13
ence (Dd ¼ dS ꢀ dR, DdH-14 ¼ ꢀ0.067).
Intracellular Rh123 accumulation assay. KB and KB/VCR
(11S)-Hydroxytetradecanoic acid. EIMS m/z [M ꢀ CH3(CH2)2 cells were seeded in six-well plates at a 1 ꢁ 106 cells density
ꢀ H2O]+ 183 (100), 143 (21), 129 (32), 95 (35), 81 (34), 73 (38), 69 and cultured for 24 h. Then fresh media containing 2.5 mg mLꢀ1
(41), 57 (29), 55 (77); HRESIMS m/z 267.1930 [M + Na]+ (calcd for of Rh123 (Sigma) and 2.5 mM of compound 2 were added and
C
14H28O3Na, 267.1931).
11-(S-MPTA)-hydroxytetradecanoic acid. 1H NMR (in of Rh123 was stopped by washing with ice-cold PBS. Cells were
pyridine-d5, 400 MHz) d 0.789 (3H, t, J ¼ 7.3 Hz, Me-14), 5.266 harvested and the intracellular mean uorescence intensity
incubated for 30 min. At the end of the time, the accumulation
(1H, m, H-11), 2.523 (2H, t, J ¼ 7.0 Hz, H-2).
(MFI) associated with Rh123 was measured with a FACS calibur
11-(R-MPTA)-hydroxytetradecanoic acid. 1H NMR (in pyri- cytometer (Ex at 485 nm Emꢀ1 at 530 nm) data analysis was
dine-d5, 400 MHz) d 0.856 (3H, t, J ¼ 7.3 Hz, Me-14), 5.264 (1H, performed using FlowJo.
m, H-11), 2.522 (2H, t, J ¼ 7.0 Hz, H-2).
P-gp expression assay. To identify the P-gp protein expres-
sion, the total protein level was measured by western blot. Cells
were seeded 1 ꢁ 106 per well in six-plates and cultured over-
night. The cells were treated with 25 mM compound 2 for
30 min, and then harvested for western blot analysis.
Sugar analysis
Compound 1 (20.0 mg) was hydrolyzed by alkali and acid, as
described in the aglycone identication section. The aqueous
phase was extracted with n-BuOH (3 ꢁ 4 mL) aer acid hydro-
lysis and concentrated to yield a colorless solid. The residue was
dissolved in H2O and directly analyzed by HPLC with authentic
samples (MeCN–H2O, 90/10): D-glucose eluted at 11.9 min,
L-rhamnose at 5.2 min. Each of these eluates was individually
collected, concentrated, and dissolved in H2O. The elutes were
identied as D-glucose [a]D21 +54.7 (c 0.1, H2O), L-rhamnose
[a]2D1 +8.7 (c 0.1, H2O) through comparisons of their specic
rotations with those of the corresponding authentic samples.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors gratefully acknowledge grants from the State Key
Laboratory of Drug Research (SIMM1601ZZ-03), the National
Natural Science Foundation of China (No. 81473111), and the
China Postdoctoral Science Foundation (No. 2016M600345).
MDR reversal assays
SRB assay. The MDR reversal activities of the test
compounds against the KB/VCR (Zhongshan School of Medi-
cine) and A549/T (KeyGEN BioTECH) cells were measured using
a sulforhodamine B (SRB) assay. Briey, cells were plated in 96-
well culture plates for 24 h and treated with serial dilutions of
vincristine (Sigma) ranging from 0.125 to 1 mM and taxel
(Selleck) ranging from 0.6 to 20 mM, with or without 25 mM of
the samples. Aer incubation for 72 h under a humidied
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