Enantioseparation and absolute configuration determination of angular-type pyranocoumarins from Peucedani radix using enzymatic hydrolysis and chiral HPLC-MS/MS analysis

Article abstract of DOI:10.3390/molecules17044236

Angular-type pyranocoumarins from Peucedani Radix (Chinese name: Qian-hu) have exhibited potential for use on treatment of cancer and pulmonary hypertension. Due to the existence of C-3′ and C-4′ chiral centers, compounds belonging to this chemical type commonly exist in enantiomers and/or diastereoisomers, which may elicit distinct activities during their interactions with the human body. In the present study, a new method, which combines enzymatic hydrolysis with chiral LC-MS/MS analysis, has been developed to determine the absolute configurations of these angular-type pyranocoumarins. Pyranocoumarins isolated from Qian-hu, their enantiomers, or metabolites were individually incubated with rat liver microsomes. As the common end product from enzymatic hydrolysis of all tested pyranocoumarins, cis-khellactone was collected and its absolute configuration was determined by comparison with (+)-cis-khellactone and (-)-cis-khellactone using chiral LC-MS/MS. The absolute configurations of all tested parent pyranocoumarins were determined by combination of LC-MS/MS, NMR and polarimetric analysis. The results revealed that the metabolite cis-khellactone retained the same absolute configurations of the stereogenic carbons as the respective parent compound. This method was proven to be rapid and sensitive and also has advantages in discriminating single enantiomers and mixtures of optical isomers with different ratios.

Full text of DOI:10.3390/molecules17044236

Molecules 2012, 17, 4236-4251; doi:10.3390/molecules17044236
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Enantioseparation and Absolute Configuration Determination
of Angular-Type Pyranocoumarins from Peucedani Radix Using
Enzymatic Hydrolysis and Chiral HPLC-MS/MS Analysis
Yue-Lin Song 1, Qing-Wen Zhang 1, Ya-Ping Li 1, Ru Yan 1,* and Yi-Tao Wang 1,2,
*
1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences,
University of Macau, Taipa, Macao SAR, 999078, China
2
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China
* Authors to whom correspondence should be addressed; E-Mails: ruyan@umac.mo (R.Y.);
ytwang@umac.mo (Y.-T.W.); Tel.: +853-8397-4876 (R.Y.); Fax: +853-2884-1358 (R.Y.);
Tel.: +853-8397-4691 (Y.-T.W.); Fax: +853-2884-1358 (Y.-T.W.).
Received: 27 February 2012; in revised form: 29 March 2012 / Accepted: 31 March 2012 /
Published: 10 April 2012
Abstract: Angular-type pyranocoumarins from Peucedani Radix (Chinese name: Qian-hu)
have exhibited potential for use on treatment of cancer and pulmonary hypertension. Due
to the existence of C-3′ and C-4′ chiral centers, compounds belonging to this chemical type
commonly exist in enantiomers and/or diastereoisomers, which may elicit distinct activities
during their interactions with the human body. In the present study, a new method, which
combines enzymatic hydrolysis with chiral LC-MS/MS analysis, has been developed
to determine the absolute configurations of these angular-type pyranocoumarins.
Pyranocoumarins isolated from Qian-hu, their enantiomers, or metabolites were
individually incubated with rat liver microsomes. As the common end product from
enzymatic hydrolysis of all tested pyranocoumarins, cis-khellactone was collected and its
absolute configuration was determined by comparison with (+)-cis-khellactone and
(−)-cis-khellactone using chiral LC-MS/MS. The absolute configurations of all tested
parent pyranocoumarins were determined by combination of LC-MS/MS, NMR and
polarimetric analysis. The results revealed that the metabolite cis-khellactone retained the
same absolute configurations of the stereogenic carbons as the respective parent
compound. This method was proven to be rapid and sensitive and also has advantages in
discriminating single enantiomers and mixtures of optical isomers with different ratios.

Molecules 2012, 17
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Keywords: angular-type pyranocoumarins; cis-khellactone; absolute configuration;
enzymatic hydrolysis; chiral LC-MS/MS
1. Introduction
Peucedani Radix (Chinese name: Qian-hu) originates from the dried roots of Peucedanum
praeruptorum DUNN, and has been used in traditional medicinal practice in China for the treatment of
cough with thick sputum and dyspnea, nonproductive cough and upper respiratory infections [1,2].
Modern pharmacological studies have revealed potent hypotensive, coronary dilatory, myocardial
protection and antitumor effects of this medicinal herb [2–5].
Angular-type pyranocoumarins, which comprise a khellactone (1, Figure 1) skeleton with C-3′ and
C-4′ substituents, were reported as the main bioactive constituents of Qian-hu [6–9]. In the Chinese
Pharmacopeia, praeruptorin A (cis-3′-angeloyl-4′-acetylkhellactone, 2) and praeruptorin B (cis-3′,4′-
diangeloylkhellactone, 3) were documented as the chemical markers for quality control of this herb
and related products [1]. Praeruptorin A and its analogues were revealed to be cytotoxic to some
cancer cells [10–12] and were able to induce apoptosis as well as reverse multidrug resistance (MDR) [5]
by suppressing expression of the efflux transporter P-glycoprotein [11]. Furthermore, the anticancer
activity of praeruptorin A has also been demonstrated in mice [12], indicating the potential of
these angular-type pyranocoumarins for cancer therapy. More interestingly, the two enantiomers of
praeruptorin A (2a and 2b, Figure 1) initiated distinct relaxant effects on isolated rat aorta rings
dependent on endothelium and nitric oxide synthesis [13], indicating the existence of stereoselectivity
in pharmacological activity of praeruptorin A.
Angular-type pyranocoumarins have two stereogenic centers (C-3′ and C-4′). Enantiomers and
diastereoisomers of this type of compounds naturally occur in some plants. In Qian-hu, the
levorotatory isomers of praeruptorin A and praeruptorin B are naturally less abundant than their
dextrorotatory enantiomers [6], indicating a chiral preference in the herb. In addition to praeruptorin A
and praeruptorin B, there are several other pyranocoumarin constituents naturally present as mixtures
of enantiomers in the herb, but so far, only (±)-praeruptorin A (2, Figure 1) has been enantioseparated
by reversed-phase and normal-phase chiral chromatography [13,14].
At present, the absolute configurations of this type of coumarin compounds are usually determined
by acidic or alkaline hydrolysis in combination of gel column chromatographic separation [6,7,15].
However, the method using acid or base hydrolysis requires large amounts of pure compounds and the
absolute configuration might be altered during acid or alkaline treatment [7,15]. Circular dichroism
(CD) has also been adopted [13,16], but the CD spectrum couldn’t distinguish the enantiomerically
enriched compound from its pure enatiomer [17]. In contrast, enzyme-mediated hydrolysis generally
breaks the ester bond without affecting the absolute configuration [18,19]. Our previous study on
metabolism of (±)-praeruptorin A by hepatic proteins from humans and rats also revealed that
cis-khellactone obtained from the levorotatory enantiomer of praeruptorin A or its antipode retained
the same absolute configuration as the parent enantiomer [14], indicating that the absolute
configuration of pyranocoumarin constituents could be determined from their hydrolytic products

Molecules 2012, 17
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formed from enzyme-mediated reactions. On the other hand, chiral LC-MS/MS analysis affords high
selectivity and sensitivity to allow the determination of drugs and metabolites at low concentrations,
thus, has been widely adopted in bioanalysis.
Figure 1. Chemical structures of pyranocoumarin compounds 1–11.
Therefore, in the present study, a method that combines enzymatic hydrolysis followed by chiral
LC-MS/MS analysis of the metabolites formed was adopted for the first time to determine the absolute
configurations of angular-type pyranocoumarin compounds from Qian-hu. The results obtained were
consolidated with data from polarimetric analysis and NMR data or by comparison with the data of the
reference compounds.

Molecules 2012, 17
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2. Results and Discussion
2.1. Identification of Pyranocoumarins from Qian-hu
By comparing the NMR, MS data and optical rotations with the literature values (Tables 1 and 2),
compounds 1–11 (Figure 1) were identified as (±)-cis-khellactone (1), (±)-praeruptorin A [(±)cis-3′-
angeloyl-4′-acetylkhellactone, 2], praeruptorin B [cis-3′,4′-diangeloylkhellactone, 3], (+)-praeruptorin
E [(+)-(3′S,4′S)-3′-angeloyl-4′-isovalerylkellactone, 4], cis-3′-acetyl-4′-angeloylkhellactone (5), cis-3′-
isovaleryl-4′-acetylkhellactone (6), (+)-cis-(3′S,4′S)-3′-angeloyl-4′-senecioylkhellactone (7), (+)-cis-
(3′S,4′S)-3′,4′-diisovalerylkhellactone (8), (−)-cis-(3′R,4′R)-3′-angeloylkhellactone (9), (−)-cis-
(3′R,4′R)-4′-angeloylkhellactone (10) and (+)-trans-(3′S,4′R)-3′-angeloylkhellactone (11), respectively.
2.2. Enantioseparation of Mixtures of Angular-Type Pyranocoumarins
Under the present analytical conditions, the enantiomers of (±)-cis-khellactone (1) achieved good
separation on a CHIRALPAK AD-RH semi-preparative column (Figure 2A). The two compounds
were eluted at the retention times of 6.7 min (1a) and 9.7 min (1b), respectively. Their optical rotations
were [α]2D0 +57° (c, 1.0) and [α]2D0 −55° (c, 1.0), which were in good agreement with those reported
previously for (+)-cis-khellactone and (−)-cis-khellactone, respectively [20]. Thus, 1a was assigned as
(+)-cis-(3′S,4′S)-khellactone and 1b as (−)-cis-(3′R,4′R)-khellactone (Figures 1 and 2A).
Enantioseparation of (±)-praeruptorin B (cis-3′,4′-diangeloylkhellactone, 3) was achieved on the
same chiral semi-preparative column using a mobile phase with a different acetonitrile/H2O composition.
The two enantiomers were eluted at 19.8 min (3a) and 22.9 min (3b), respectively. The optical
rotations measured [3a, [α]2D0 +37° (c, 1.0); 3b, [α]2D0 −36° (c, 1.0)] for the enantiomers were similar to
the data reported by Okuyama and co-workers [7]. Thus the two enantiomers were identified as
(+)-praeruptorin
B
[(+)-cis-(3′S,4′S)-3′,4′-diangeloylkhellactone, 3a] and (−)-praeruptoin
B
[(−)-cis-(3′R, 4′R)-3′,4′-diangeloylkhellactone, 3b] (Figures 1 and 2B).
2.3. Determination of Absolute Configuration of Angular-Type Pyranocoumarins
Our previous studies [14,21] and Ruan’s report [22] on the metabolisms of (±)-praeruptorin A (2),
(+)-praeruptorin A (2a), (−)-praeruptorin A (2b), (+)-praeruptorin B (3a) and (+)-praeruptorin E (4)
revealed that all these angular-type pyranocoumarins underwent stepwise hydrolysis and generated
cis-khellactone without alteration of the absolute configuration when they were incubated with liver
microsomal proteins from rats or humans in the presence of a NADPH-regenerating system. This
finding suggests that the absolute configurations of this type of coumarins can be determined from the
configurations of C-3′ and C-4′ of khellactone produced from hydrolysis of the parent coumarins by rat
or human hepatic phase I isozymes.

Molecules 2012, 17
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Table 1. HPLC-MS/MS data and optical rotations of pyranocoumarins from Qian-hu and their hydrolytic products.
compound
MS1
MW
MS2
Optical rotation (c Hydrolytic metabolite Identity
1.0, CDCl3)
0°
(+/− cis-khellactone)
1
263[M+H]+; 285[M+Na]+
263[M+H]+; 285[M+Na]+
263[M+H]+; 285[M+Na]+
262
262
262
245,203
245,203
245,203
+:− = 1:1
(±)-cis-khellactone
1a
1b
2
−55°
-
(−)-cis-(3′S, 4′S)-khellactone
+57°
+
(+)-cis-(3′R, 4′R)-khellactone
404[M+NH4]+; 409[M+Na]+ 386
404[M+NH4]+; 409[M+Na]+ 386
404[M+NH4]+; 409[M+Na]+ 386
444[M+NH4]+; 449[M+Na]+ 426
444[M+NH4]+; 449[M+Na]+ 426
444[M+NH4]+; 449[M+Na]+ 426
446[M+NH4]+; 451[M+Na]+ 428
404[M+NH4]+; 409[M+Na]+ 386
404[M+NH4]+; 409[M+Na]+ 386
406[M+NH4]+; 411[M+Na]+ 388
406[M+NH4]+; 411[M+Na]+ 388
444[M+NH4]+; 449[M+Na]+ 426
448[M+NH4]+; 453[M+Na]+ 430
327,245,227 0°
+:− = 1:1
(±)-cis-3′-angeloyl-4′-acetylkhellactone
(+)-cis-(3′S, 4′S)-3′-angeloyl-4′-acetylkhellactone
(−)-cis-(3′R, 4′R)-3′-angeloyl-4′-acetylkhellactone
cis-3′, 4′-diangeloxylkhellactone
2a
2b
3
327,245,227 +59°
327,245,227 −61°
327,245,227 +3.4°
327,245,227 +36°
327,245,227 −37°
327,245,227 +35°
309,245,227 +2.9°
309,245,227 +3.5°
329,245,227 +27°
329,245,227 +33°
327,245,227 +31°
329,245,227 +35°
267,245,227 −57°
267,245,227 −39°
327,245,227 +11°
-
+
+:− = 2:3
3a
3b
4
-
(+)-cis-(3′S, 4′S)-3′, 4′-diangeloxylkhellactone
(−)-cis-(3′R, 4′R)-3′, 4′-diangeloxylkhellactone
(+)-cis-(3′S, 4′S)-3′-angeloxyl-4′-isovalerylkhellactone
cis-3′-acetyl-4′-angeloylkhellactone
+
-
5
+:− = 7:1
5a
6
+
(+)-cis-(3′R, 4′R)-3′-acetyl-4′-angeloylkhellactone
cis-3′-isovaleryl,4′-acetylkhellactone
+:− = 1:8
6a
7
-
(+)-cis-(3′S, 4′S)-3′-isovaleryl,4′-acetylkhellactone
(+)-cis-(3′S, 4′S)-3′-angeloyl-4′-senecioylkhellactone
(+)-cis-(3′S, 4′S)-3′,4′-diisovalerylkhellactone
(−)-cis-(3′R, 4′R)-3′-angeloylkhellactone
(−)-cis-(3′R, 4′R)-4′-angeloylkhellactone
(+)-trans-(3′S, 4′R)-3′-angeloylkhellactone
-
8
-
9
367[M+Na]+; 383[M+K]+
367[M+Na]+; 383[M+K]+
367[M+Na]+; 383[M+K]+
344
344
344
+
10
11
+
N.A.
N.A.: not applicable.

Molecules 2012, 17
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Table 2. 1H-NMR spectroscopic data of angular-type pyranocoumarin compounds 1-11 in CDCl3 (600 M).
Compound
Position
1
2
3
4
5
6
7
8
9
10
11
3
6.27 (d, 9.5) 6.26 (d, 9.5) 6.23 (d, 9.5) 6.25 (d, 9.6) 6.24 (d, 9.6) 6.25 (d, 9.6) 6.23 (d, 9.5) 6.24 (d, 9.5) 6.27 (d, 9.6) 6.24 (d, 9.5) 6.27 (d, 9.5)
7.67 (d, 9.5) 7.61 (d, 9.5) 7.60 (d, 9.5) 7.60 (d, 9.6) 7.60 (d, 9.6) 7.61 (d, 9.6) 7.59 (d, 9.5) 7.60 (d, 9.5) 7.65 (d, 9.6) 7.61 (d, 9.5) 7.67 (d, 9.5)
7.34 (d, 8.7) 7.37 (d, 8.6) 7.37 (d, 8.6) 7.35 (d, 8.6) 7.37 (d, 8.6) 7.36 (d, 8.6) 7.36 (d, 8.6) 7.36 (d, 8.6) 7.36 (d, 8.5) 7.38 (d, 8.7) 7.35 (d, 8.6)
6.81 (d, 8.7) 6.82 (d, 8.6) 6.82 (d, 8.6) 6.81 (d, 8.6) 6.82 (d, 8.6) 6.81 (d, 8.6) 6.81 (d, 8.6) 6.81 (d, 8.6) 6.81 (d, 8.5) 6.82 (d, 8.7) 6.83 (d, 8.6)
3.89 (d, 5.0) 5.37 (d, 5.0) 5.46 (d, 5.0) 5.41 (d, 4.8) 5.37 (d, 4.8) 5.34 (d, 5.0) 5.42 (d, 4.8) 5.34 (d, 4.8) 5.49 (d, 4.8) 4.10 (d, 4.8) 5.28 (d, 3.6)
5.23 (d, 5.0) 6.65 (d, 5.0) 6.72 (d, 5.0) 6.63 (d, 4.8) 6.65 (d, 4.8) 6.57 (d, 5.0) 6.67 (d, 4.8) 6.57 (d, 4.8) 5.25 (d, 4.8) 6.51 (d, 4.8) 5.09 (d, 3.6)
4
6
7
3′
4′
5′
6′
2
1.42 (s)
1.48 (s)
-
1.45 (s)
1.49 (s)
-
1.47 (s)
1.51 (s)
-
1.46 (s)
1.49 (s)
-
1.45 (s)
1.48 (s)
-
1.42 (s)
1.46 (s)
2.26 (m);
2.25 (m)
2.10 (m)
1.46 (s)
1.50 (s)
-
1.45 (s)
1.47 (s)
2.30 (m);
2.20 (m)
1.45 (s)
1.51 (s)
-
1.45 (s)
1.50 (s)
-
1.42 (s)
1.52 (s)
-
3′′
4′′
5′′
2′′′
-
-
-
-
6.14 (q, 7.2) 6.13 (q, 7.2) 6.13 (q, 7.2) 2.11 (s)
6.13 (q, 7.2) 2.15 (m)
6.18 (q, 7.2)
-
-
-
-
6.11 (q, 7.2)
1.92 (d, 7.2)
1.85 (s)
-
1.97 (d, 7.2) 2.00 (d, 7.2) 1.98 (d, 7.2)
-
-
-
0.98 (d, 7.2) 1.98 (d, 7.2) 0.98 (d, 7.2) 2.00 (d, 7.2)
0.98 (d, 7.2) 1.86 (s) 0.98 (d, 7.2) 1.91 (s)
1.88 (s)
2.12 (s)
1.87 (s)
-
1.89 (s)
2.28 (m);
2.20 (m)
2.15 (s)
5.63 (s)
2.30 (m);
2.20 (m)
2.15 (m)
0.98, d, 7.2
0.98, d, 7.2
[26,27]
-
3′′′
-
-
7.25 (q, 7.2) 2.14 (m)
7.26 (q, 7.2)
-
-
-
6.13 q, 7.2
2.02, d, 7.2
1.91, s
-
4′′′
-
-
1.98 (d, 7.2) 0.97 (d, 7.2) 2.01 (d, 7.2)
-
1.89 (s)
2.20 (s)
[26]
-
-
5′′′
-
-
1.85 (s)
[6]
0.97 (d, 7.2) 1.88 (s)
[8] [23]
-
-
-
Ref.
[6]
[6]
[24,25]
[14]
[14]
[20]

Molecules 2012, 17
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Figure 2. Typical HPLC-UV (323 nm) chromatograms of compounds 1 (A) and 3 (B) on a
CHIRALPAK AD-RH semi-preparative column.
To confirm this speculation, (+)-praeruptorin A [[α]2D0 +59° (c, 1.0), 2a], (−)-praeruptorin A
[[α]2D0 −61° (c, 1.0), 2b], (+)-praeruptorin E (4), and the two metabolites of 2b formed in rat plasma,
(−)-cis-(3′R,4′R)-3′-angeloyl khellactone [[α]2D0 −57° (c, 1.0
, 9] and (−)-cis-(3′R,4′R)-4′-angeloyl-
)
khellactone [[α]2D0 −39° (c, 1.0), 10], were incubated with rat liver microsomes. The chemical structures
and absolute configurations of 2a, 2b, 9 and 10 have been unambiguously identified using NMR
analysis and optical rotation data in our laboratory [14], while 4 was commercially available and its
chemical structure was double checked by comparing its mass spectral profile and optical rotation data
[[α]2D0 +35° (c, 1.0)] with the literature report [8].
When incubated with rat liver microsomes (RLMs) in presence of the NADPH-regenerating system,
all these five pyranocoumarins generated cis-khellactone which was eluted at 12.0 min under present
achiral HPLC conditions and exhibited identical UV and MS spectra to those of the authentic
(±)-cis-khellactone (Figure 3). When the cis-khellactone produced from each reaction was collected
and analyzed using chiral LC-MS/MS, the products formed from (+)-praeruptorin E (4) and
(+)-praeruptorin A (2a) exhibited the same retention time at 9.0 min as that of (−)-cis-khellactone
(1b, Figure 4A), while those metabolites of (−)-praeruptorin A (2b), (−)-(3′R, 4′R)-3′-
angeloylkhellactone (9) and (−)-(3′R,4′R)-4′-angeloylkhellactone (10) shared the same retention time
(10.6 min) with (+)-cis-khellactone (1a, Figure 4B).
These results together with the optical rotation data of these parent pyranocoumarins (Table 1)
support that their respective hydrolytic metabolite cis-khellactone keeps the absolute configurations
during hydrolysis of the parent compounds in rat liver microsomal proteins.

Molecules 2012, 17
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Figure 3. Typical achiral HPLC-UV (323 nm) chromatograms of incubations of compounds
2–10 in rat liver microsomes in the presence of a NADPH-regenerating system.

Molecules 2012, 17
Figure 4. Typical extracted ion current chromatograms (XIC) (263.1 > 203.1) of authentic
4244
1a (A) and 1b (B), and samples from incubations of compound 5 (C) and 6 (D) with rat
liver microsomes under chiral LC-MS/MS analytical conditions.
In contrast, when 2a [(+)-cis-(3′S,4′S)-3′-angeloyl-4′-acetylkhellactone] was subjected to basic
hydrolysis [20], the formation of compound 11 [(+)-trans-(3′S,4′R)-3′-angeloylkhellactone] was
evidenced by NMR and LC-MS/MS analysis and its optical rotation (Tables 1 and 2).
Peucedanocoumarins I, II and III, which are also trans-khellactone type coumarins, yielded
cis-khellactone after alkaline hydrolysis [23]. Thus, epimerization might occur during alkaline
treatment of pyranocoumarins and finally may cause misidentification of the absolute configurations of
parent compounds.
This method was further applied to the determination of the absolute configuration of another four
pyranocoumarin compounds 5a, 6a, 7 and 8 isolated from Qian-hu. cis-Khellactone obtained from the
enzymatic hydrolysis of compound 5a was eluted at 12.0 min on the achiral column (Figure 3) and
only appeared at 10.6 min on the chiral LC-MS/MS (Figure 4B), and identified as (+)-cis-khellactone
by comparison with the reference compound. Thus a (3′R,4′R) configuration of compound 5a was
suggested. Furthermore, the optical rotation of 5a in chloroform was [α]2D0 +3.5° (c, 1.0) and the NMR
data supported the structure as cis-3′-acetyl-4′-angeloylkhellactone (Table 2). These findings
demonstrated that the absolute configuration of 5a predicted from the absolute configuration of its
metabolite cis-khellactone corresponded well with the structure identified based on LC-MS/MS, NMR
and polarimetric analysis (Tables 1 and 2). These data of 5a were in good agreement with those of
pteryxin [(3′R, 4′R)-3′-acetyl-4′-angeloylkhellactone], which has been isolated from P. praeruptorum [23],
thus, compound 5a was unambiguously identified as pteryxin [(+)-cis-(3′R,4′R)-3′-acetyl-4′-angeloyl-
khellactone].

Molecules 2012, 17
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Similarly, the end products obtained from hydrolysis of compounds 6a, 7, 8 by RLMs (Figure 3)
showed the same retention time as that of (-)-cis-khellactone on the chiral LC-MS/MS system (Figure
4A), supporting the existence of cis-(3′S,4′S) configurations in these compounds. Correspondingly, the
NMR data (Table 2) of compound 6a supported it as cis-3′-isovaleryl-4′-acetyl khellactone and
polarimetric measurement revealed a positive optical rotation [[α]2D0 +33° (c, 1.0)]. Compound 6a was
thereby identified as (+)-cis-(3′S,4′S)-3′-isovaleryl-4′-acetylkhellactone. NMR analysis (Table 2) of
compounds 7 and 8 identified their structures as cis-3′-angeloyl-4′-senecioylkhellactone and cis-3′,4′-
diisovalerylkhellactone, respectively. Their optical rotations were +31° (c, 1.0) and +35° (c, 1.0).
Taken together, the structures were assigned as (+)-cis-(3′S,4′S)-3′-angeloyl-4′-senecioylkhellactone (7)
and (+)-cis-(3′S, 4′S)-3′,4′-diisovalerylkhellactone (8). So far, (±)-dihydrosamidin, the racemic form of
compound 6a, has been isolated from P. Turgeniifolium [24,25], while neither the racemate nor any of
the single enantiomers has been reported for Qian-hu. Both 3′-angeloyl-4′-senecioylkhellactone and
3′,4′-diisovalerylkhellactone have been reported in P. japonicum, but not in P. praeruptorum (Qian-hu) [26]
and their absolute configurations were not determined in the previous report [27]. Again, determination
of the absolute configurations of pyranocoumarin compounds 5a, 6a, 7 and 8 based on chiral LC-MS/MS
analysis of their respective hydrolytic metabolite cis-khellactone formed by RLMs were proved to be
feasible and should be applicable to other pyranocoumarins that have a hidden C-3' and/or C-4'
hydroxy functionality that can be released by enzymatic transformations.
It’s interesting to note that compounds 5 and 6, which were obtained from Qian-hu to prepare 5a
and 6a through crystallization, exhibited similar positive optical rotation and identical NMR data as
compounds 5a and 6a, respectively. However, when subjected to enzymatic hydrolysis, both
compounds formed the two optical isomers of cis-khellactone (Figure 4C,D), indicating that
compounds 5 and 6 were isolated from Qian-hu as enantiomerically enriched compounds. This was
further confirmed by two peaks observed in chromatograms obtained from enantioseperation of
compounds 5 and 6 (data not shown) and the enantiometric ratios (±) of cis-khellactone formed from 5
(7/1) and 6 (1/8) by RLMs (Table 1). Thus, the NMR data plus net optical rotation could not
distinguish the enantiomerically enriched angular-type pyranocoumarin from its pure enantiomer.
In the present study, (±)-praeruptorin B (3) was enantioseparated, for the first time, to obtain the two
enantiomers. The two optical isomers (3a and 3b) were also subjected to enzymatic hydrolysis
followed by chiral LC-MS/MS analysis individually. Similar to those observed with the two isomers
from (±)-praeruptorin A (2), cis-khellactone from (+)-praeruptorin B (3a) shared the same retention
time (9.0 min) with (−)-cis-khellactone (Figure 4A), while cis-khellactone from (−)-praeruptorin B (3b)
was observed at 10.6 min, the same as (+)-cis-khellactone (Figure 4B), suggesting that the metabolite
cis-khellactone keep the absolute configuration as their respective parent optical isomer. Again, this
result agreed well with that obtained from a combination of NMR and polarimetric analysis (Tables 1
and 2) in the present study and the previous report [7]. Thus, (+)-praeruptorin B (3a) was identified as
(+)-cis-(3′S,4′S)-3′,4′-diangeloylkhellactone and (−)-praeruptorin B (3b) as (−)-cis-(3′R,4′R)-3′,4′-
diangeloylkhellactone.
The stereoselectivity in pharmacological actions [13], metabolism [14] and absorption [28] of
praeruptorin A have been revealed by previous studies, indicating that it is of great importance to
determine the absolute configurations of the angular-type pyranocoumarins when correlate their
structures with the resultant biological interactions during pharmacological/toxicological evaluation

Molecules 2012, 17
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and/or ADME screening. Furthermore, because most pyranocoumarins from Qian-hu naturally exist as
enantiomers and/or diastereoisomers, misidentification of the identity of the compound studied has
occurred [29–31]. Thus, it is crucial to establish a method which enables rapid determination of the
absolute configurations of the pyranocoumarin compounds to be worked on. These findings obtained
in the present study demonstrated that it is feasible to determine the absolute configurations of
pyranocoumarins from that of the hydrolytic product khellactone using enzymatic hydrolysis coupled with
chiral LC-MS/MS analysis. Because of the high sensitivity and selectivity afforded by LC-MS/MS
techniques, this method can be used to determine the configuration of compounds of this chemical type
with advantages in lower quantity of the tested compounds required for in vitro incubation.
3. Experimental
3.1. Materials
Qian-hu was obtained from Ningguo, Anhui Province, China and the crude drug was authenticated
as the dried roots of P. praeruptorum DUNN by Professor Pengfei Tu from Department of Natural
Medicines, Peking University (Beijing, China). The specimens were stored at the State Key Laboratory
of Quality Research in Chinese Medicine, University of Macau.
The crude drug (6 kg) was crushed into a powder and extracted with 95% ethanol (50 L × 3, 80 °C)
to afford an extract (1.83 kg). An aliquot of the extract (50 g) was loaded onto a silica gel column
(200–300 mesh, 800 g) and fractionated with petroleum ether-ethyl acetate (20:1→1:2, v/v) to get 11
fractions (Fractions 1–11). Fraction 4 (4.2 g) was loaded onto another silica gel column (200–300 mesh,
100 g) was and compound 2 (432 mg) was eluted with petroleum ether-ethyl acetate (8:1→1:1, v/v).
Compounds 3 (195 mg) and 4 (52 mg) were obtained by recrystallization of Fraction 5 (5.3 g) and
Fraction 8 (7.1 g) from MeOH, respectively. Compound 6 (12 mg) was isolated from the remaining
solution of Fraction 5 using a semi-preparative COSMOSIL 5C18-AR-II column (250 mm × 10.0 mm
i.d., particle size 5 µm, Nacalai Tesque, Kyoto, Japan) that eluted with MeOH–H2O (65:35, v/v) at a
flow rate of 2.5 mL/min. The remaining solution of Fraction 8 was introduced to the same
semi-preparative HPLC system and eluted with MeOH-H2O (69:31, v/v) to afford 7 (11 mg) and 8 (17 mg).
Compound 5 (13 mg) was prepared from Fraction 3 by semi-preparative HPLC with an isocratic
elution utilizing MeOH-H2O (77:23, v/v). Compounds 5a [(+)-cis-(3′R,4′R)-3′-acetyl-4′-angeloyl-
khellactone] and 6a [(+)-cis-(3′S,4′S)-3′-isovaleryl-4′-acetylkhellactone] were obtained as white
needles by recrystallization of 5 and 6 from methanol.
A basic hydrolysis of 2 (70 mg) was carried out to obtain 1 (23 mg) following a procedure
described previously by our group [14]. (+)-Praeruptorin A [(+)-cis-(3′S,4′S)-3′-angeloyl-4′-acetyl-
khellactone, 2a] and (−)-praeruptorin A [(−)-cis-(3′R,4′R)-3′-angeloyl-4′-acetylkhellactone, 2b] were
obtained by enantioseperation of compound 2 according to our previous report [14]. (−)-cis-(3′R,4′R)-
3′-angeloylkhellactone (9) and (−)-cis-(3′R,4′R)-4′-angeloylkhellactone (10) were prepared from an
incubation of compound 2b with rat plasma and unambiguously identified using NMR and LC-MS/MS
analysis [14]. Compound 11 was obtained from basic hydrolysis of compound 2a, according to a
method reported by Wu and co-workers [20]. Additionally, authentic compounds 2, 2a and 4 were
purchased from Shanghai Traditional Chinese Medicine Research Center (Shanghai, China) and their

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identities were confirmed using LC-MS/MS and polarimetric analysis. The purity of all the compounds
was all above 98% as determined by achiral HPLC-UV. The identities of all pyranocoumarin
constituents 1–11 isolated from Qian-hu were identified using polarimetric analsyis, LC-UV-MS/MS
and NMR analysis and confirmed by comparison with previous reports. The spectrometric and
spectroscopic data were summarized in Tables 1 and 2.
Pooled rat liver microsomes (RLMs) were prepared at the School of Biomedical Sciences, the
Chinese University of Hong Kong by differential centrifugation according to a standard procedure
reported previously [32]. The content of microsomal proteins was determined using Lowry’s
method [33] and stored at −80 °C until use.
Glucose 6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PD), and β-nicotinamide
adenine dinucleotide phosphate (β-NADP+) were obtained from Sigma-Aldrich Corp. (St. Louis, MO,
USA). Formic acid, acetonitrile and methanol of HPLC grade were purchased from Merck (Darmstadt.
Germany). Ultra-pure water was obtained in house using a Milli-Q plus water purification system
(Millipore, Bedford, MA, USA).
3.2. Enantioseparation of Angular-type Pyranocoumarins from Qian-hu
Enantioseparation of the enantiometric mixtures (±)-cis-khellactone (1) and (±)-praeruptorin B (3)
were carried out on a semi-preparative CHIRALPAK AD-RH column (10 × 250 mm I.D., particle size
5 µm, Daicel, Tokyo, Japan). The mobile phases were acetonitrile-H2O with a volumetric ratio of
30/70 for 1 and 45/55 for 3. The flow rates were 2.5 mL/min for elution of both analytes. Each analyte
eluted was collected separately and the solvent was removed under reduced pressure to yield 1a (9.5 mg)
and 1b (8.5 mg), 3a (23.6 mg) and 3b (19.9 mg). The purity of all the enantiomers was more than 98%
as determined using chiral LC-UV/MS/MS.
3.3. Polarimetric and NMR Analysis
The polarimetric analysis was performed on a Perkin-Elmer 243B digital polarimeter
(PerkinElmer, Netherlands) in chloroform at 589.3 nm, 20 °C. Compounds 1–11 were dissolved in
1
CDCl3 containing 0.3% trimethylsilane (TMS) and their H NMR spectra were acquired on a Bruker
Avance 600 spectrometer (600 MHz, Bruker GmbH, Bremen, Germany). Chemical shifts were
reported in δ scale in units of part per million (ppm) with TMS as the reference, and coupling constants
(J) were expressed in units of Hertz (Hz).
3.4. Hydrolysis of Angular-type Pyranocoumarins from Qian-hu by Rat Liver Microsomes
Enzymatic hydrolysis of each of the angular-type pyranocoumarins (final concentration 25 µM) was
carried out in a 200 µL reaction system containing 1 mg/mL of rat liver microsomal proteins in
0.1 M potassium phosphate buffer (pH 7.4) at 37 °C. The reactions were initiated by adding the
NADPH-regenerating system (4 mM MgCl2, 1 mM β-NADP+, 1 mM G-6-P and 1 U/mL of G-6-PD).
Reactions were stopped at 60 min by adding an equal volume of ice-cold methanol and the samples
centrifuged to remove the proteins before subjected to achiral HPLC-UV analysis.

Molecules 2012, 17
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3.5. Achiral HPLC-UV Analysis
The achiral HPLC-UV analysis was performed on an Agilent 1200 series liquid chromatographic
system (Agilent Technologies, Palo Alto, CA, USA) that equipped with a vacuum degasser, a
quaternary pump, an autosampler and a diode array detector (DAD) system. Data acquisition was
controlled by an Agilent ChemStation B 3.0 software. Sample separation was performed on an ODS
reversed-phase C18 column (250 mm × 4.6 mm I.D., particle size 5 µm, Agilent) at 35 °C. The mobile
phase consisted of water containing 0.1% formic acid (A) and methanol (B). A gradient elution was
carried out at 1.0 mL/min as follows: 0–4 min, 25–50% B; 4–16 min, 50–53% B; 16–17 min, 53–56% B;
17–20 min, 56% B; 20–23 min, 56–69% B; 23–40 min, 69–76% B; 40–41 min, 76–100% B; 41–52 min,
100% B. All reference compounds and samples from reactions were monitored at 323 nm and UV
spectra were recorded over 200–400 nm. The injection volume was 70 µL. cis-Khellactone, the end
product yielded from the aforementioned reactions of all pyranocoumarins, was eluted at the retention
time of 12.0 min under the present analytical conditions and collected separately for further chiral
LC-MS/MS analysis.
3.6. Chiral HPLC-MS/MS Analysis
Chiral LC-MS/MS analysis was performed on an Agilent 1200SL series (Agilent) liquid
chromatography coupled online with an API 4000 Q-trap@ mass spectrometer (Applied Biosystems,
Foster City, CA, USA). The LC system consisted of a vacuum degasser, a binary pump, and an
autosampler. Chromatographic separation was performed on an analytical CHIRALPAK AD-RH
column (5 × 150 mm I.D., particle size 5 µm, Daicel, Tokyo, Japan), which was eluted with
acetonitrile-H2O (30:70, v/v) at a flow rate of 0.65 mL/min. The mass spectrometer was equipped with
a Turbo VTM source and a Turbo Ion Spray probe (500 °C) and operated in positive ion multiple
reactions monitoring (MRM) mode. Ion optics was tuned using polypropylene glycol (PPG) standard
dilution solvents. Nitrogen was used as the nebulizer, heater, curtain and collision gases. Optimum ion
source parameters for cis-khellactone were as follows: nebulizer (GS1), heater (GS2) and curtain gas
flow rates 50, 50, 10 instrument units, respectively; ionspray voltage 4500 V; heater gas temperature
550 °C; declustering potential (DP) 100 V. The ion pairs used for monitoring cis-khellactone were
263.1 > 245.1 and 263.1 > 203.1. Collision energies (CE) were set as 25 eV and 30 eV for the two ion
pairs, respectively. The Applied Biosystems Analyst Software package (Version 1.5) was used for
instrument control, data acquisition and processing.
4. Conclusions
In the present study, a new method using enzymatic hydrolysis coupled with chiral LC-MS/MS
analysis was developed to assist the determination of the absolute configuration of angular-type
pyranocoumarins. It has been proved to be rapid and sensitive and demonstrated success in
identification of this chemical type from Qian-hu.

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Acknowledgments
The research was supported by the National Basic Research Program of China 973 program
(Grant No.2009CB522707) and the Research Committee of University of Macau (reference
No.UL016/09-Y4/CMS/WYT01/ICMS, MYRG 208 (Y2-L4)-ICMS11-WYT).
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Sample Availability: Samples of compounds 1–11 and enantiomers of compounds 1–3 are available
from the authors.
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