The Gastrointestinal Tract Metabolism and Pharmacological Activities of Grosvenorine, a Major and Characteristic Flavonoid in the Fruits of Siraitia grosvenorii

Article abstract of DOI:10.1002/cbdv.201400397

Grosvenorine is the major flavonoid compound of the fruits of Siraitia grosvenorii (Swingle) C. Jeffrey, a medical plant endemic to China. In the present study, for the first time, the grosvenorine metabolism in an in vitro simulated human gastrointestinal tract (including artificial gastric juice, artificial intestinal juice and intestinal flora), as well as its pharmacological activities (including anti-complement, antibacterial and antioxidant activities), was investigated. The results showed that grosvenorine was metabolized by human intestinal flora; its four metabolites were isolated by semi-preparative HPLC and identified by NMR as kaempferitrin, afzelin, α-rhamnoisorobin, and kaempferol. Further pharmacological evaluation showed that grosvenorine exhibited good antibacterial and antioxidant activities, with its metabolites possessing more potent activities. Although grosvenorine did not present obvious anticomplement activity, its metabolites showed interesting activities. This study revealed that intestinal bacteria play an important role in the gastrointestinal metabolism of grosvenorine and significantly affect its pharmacological activities.

Full text of DOI:10.1002/cbdv.201400397

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CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
The Gastrointestinal Tract Metabolism and Pharmacological Activities of
Grosvenorine, a Major and Characteristic Flavonoid in the Fruits of Siraitia
grosvenorii
by Mengyue Wang1), Shihua Xing1), Thithanhthuy Luu, Min Fan, and Xiaobo Li*
School of Pharmacy, Shanghai Jiao Tong University, No. 800 Dongchuan Road, Minhang District,
Shanghai 200240, P. R. China
(phone: þ86-21-34204806; fax: þ86-21-34204804; e-mail: xbli@sjtu.edu.cn)
Grosvenorine is the major flavonoid compound of the fruits of Siraitia grosvenorii (Swingle)
C. Jeffrey, a medical plant endemic to China. In the present study, for the first time, the grosvenorine
metabolism in an in vitro simulated human gastrointestinal tract (including artificial gastric juice,
artificial intestinal juice and intestinal flora), as well as its pharmacological activities (including anti-
complement, antibacterial and antioxidant activities), was investigated. The results showed that
grosvenorine was metabolized by human intestinal flora; its four metabolites were isolated by semi-
preparative HPLC and identified by NMR as kaempferitrin, afzelin, a-rhamnoisorobin, and kaempferol.
Further pharmacological evaluation showed that grosvenorine exhibited good antibacterial and
antioxidant activities, with its metabolites possessing more potent activities. Although grosvenorine
did not present obvious anticomplement activity, its metabolites showed interesting activities. This study
revealed that intestinal bacteria play an important role in the gastrointestinal metabolism of
grosvenorine and significantly affect its pharmacological activities.
Introduction. – Siraitia grosvenorii (Swingle) C. Jeffrey (Momordica grosvenorii
Swingle), an endemic Chinese medical plant, grows principally in Guangxi Province,
where it has been cultivated for more than 200 years [1]. Its ripe fruit, Siraitiae Fructus
(Chinese Luo-han-guo) is recorded in the Chinese Pharmacopoeia and it is commonly
used as a traditional Chinese medicine (TCM) to clinically treat pharyngolaryngitis,
bronchitis, asthma, and diabetes by oral administration [2]. Siraitiae Fructus was also
listed as a medicinal and edible species by the Chinese Ministry of Health in 1987.
Currently, it is being used as a natural sweetener because of its good safety, high
sweetness, and excellent mouthfeel. According to the report, the fruit of S. grosvenorii
has obvious antibacterial, anti-oxidative, antitussive, anti-asthmatic, liver-protective,
glucose-lowering, immune-regulatory, anticancer, and anti-inflammatory activities [3].
The primary chemical constituents of Siraitiae Fructus are cucurbitane glycosides and
flavonoids, of which grosvenorine is the main flavonoid glycoside compound and was
found to exist only in Siraitiae Fructus [4][5]. Unfortunately, there are no reports
regarding the pharmacological activities and metabolism of grosvenorine.
The majority of TCMs, including Siraitiae Fructus, are orally administered, so their
components inevitably come into contact with gastric juice, intestinal juice, and
1
)
These authors contributed equally to this work.
ꢀ 2015 Verlag Helvetica Chimica Acta AG, Zürich

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bacteria flora in the gastrointestinal tract. Therefore, oral TCMs are often metabolized
in the gastrointestinal tract due to the low gastric pH conditions, and because of the
presence of digestive enzymes and intestinal bacteria, many of the chemical
components, such as flavonoids, triterpenes, lignanoids, and phenolics, are transformed
prior to absorption [6]. These transformations can modulate their biological activities
and toxicities, and, in most cases, the benefits of oral TCMs should be attributed to their
bioactive metabolites produced by the gastrointestinal tract. Thus, it is important to
study the metabolism of oral TCMs in the gastrointestinal tract to better characterize
their biological effects in vivo [7][8].
Siraitiae Fructus is a fruit rich in flavonoids [9]. The available literature strongly
supports that flavonoids from plants act as anticomplement and antioxidant agents [10–
13]. Additionally, flavonoids have been reported to possess antimicrobial activity
against many pathogenic bacteria, including Staphylococcus aureus, Bacillus subtilis,
Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli, and Salmonella typhi
[14][15]. Recently, the novel flavonoid siraitiflavandiol, as derived from Siraitiae
Fructus, was found to inhibit the growth of the oral bacterial species Streptococcus
mutans and Porphyromonas gingivalis, as well as the yeast Candida albicans [16].
However, studies on the antibacterial, anticomplement, and antioxidant capabilities of
grosvenorine have not been performed.
The aim of this study was to elucidate the pharmacological activities and
metabolism of grosvenorine, a major flavonoid from the fruits of S. grosvenorii, in a
simulated human gastrointestinal tract. Grosvenorine was anaerobically incubated with
artificial gastric juice, artificial intestinal juice, and intestinal flora in vitro, and its
metabolites were isolated and identified. The antibacterial, anticomplement, and
antioxidant activities of grosvenorine and its metabolites were also tested.
Results and Discussion. – 1. Preparation of Grosvenorine. To ensure a sufficient
amount of grosvenorine for the metabolic and pharmacological experiments, 370 mg of
grosvenorine, with a purity of 98.3% as determined by HPLC, were purified from the
H2O extract of Siraitiae Fructus by repeated column chromatography. Because the
1
complete H- and 13C-NMR spectra of grosvenorine have not been reported, we
attempted to assign all of the chemical shifts with the aid of the 2D-NMR spectra
(1H,1H-COSY, HSQC, and HMBC). The spectral data of grosvenorine were assigned
and are listed in the Exper. Part.
2. Investigation of Metabolism in a Simulated Gastrointestinal Environment. After
incubation with artificial gastric and intestinal juices at 378 for 24 h, the peak areas of
the HPLC-DAD chromatograms of grosvenorine were almost unchanged at different
incubation times (RSD <3.0%, n¼5), and no metabolites were observed. However,
significant changes were observed after incubation with human fecal microflora.
Grosvenorine was almost completely transformed after a 12 h incubation, and four
metabolites (M1, M2, M3, and M4) were found (Fig. 1). The results suggest that
grosvenorine is primarily metabolized in the colon, and intestinal bacteria play an
important role in its gastrointestinal metabolism.
3. Isolation and Identification of the Grosvenorine Metabolites Produced by
Intestinal Bacteria. To investigate the metabolites produced by human intestinal
bacteria, grosvenorine (300 mg) was anaerobically incubated with human fecal

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Fig. 1. The analytic HPLC profile of grosvenorine and the metabolites produced by human intestinal
bacteria. G: grosvenorine; M1–M4: metabolites. Chromatographic column: Athena C18-WP column
(4.6Â250 mm, 5 mm). Column temperature: 308. Detection wavelength: 280 nm. Injection volume:
10 ml. Eluent: 0.1% HCOOH soln. and MeCN; MeCN linear gradient increasing from 10% MeCN to
100% over a 40-min period. Flow rate: 1.0 ml/min.
microflora for 6 h, after which the metabolites were extracted with AcOEt. The extract
was evaporated under reduced pressure to produce a residue (0.52 g). Based on the
analytic HPLC results (Fig. 1), the residue was directionally isolated by semi-prep.
HPLC under the same conditions as the analytic HPLC, with the exception that the
column was a prep. column, and the flow rate was 3.0 ml/min. Here, four metabolites
(M1 (64 mg), M2 (19 mg), M3 (13 mg), and M4 (5 mg)) were isolated.
The metabolites M1, M2, M3, and M4 were identified as kaempferitrin [17], afzelin
[18], a-rhamnoisorobin [19], and kaempferol [20], resp., by NMR analysis. Although
kaempferitrin is a common flavonoid glycoside widely distributed in herbs, the assigned
chemical shifts (in (D6)DMSO) of the C-atoms of two rhamnosyls, C2 and C9 of
aglycone, are often inconsistent in the literature. Thus, to correctly assign these
chemical shifts, the 2D-NMR spectra (1H,1H-COSY, HSQC, and HMBC) were
determined, and the spectral data were listed in the Exper. Part.
The time course of the transformation of grosvenorine is shown in Fig. 2.
Grosvenorine was quickly converted to kaempferitrin by human intestinal bacteria.
Kaempferitrin was further metabolized by the removal of the respective rhamnose
units to produce afzelin and a-rhamnoisorobin, and then finally converted to
kaempferol. The metabolic rate of a-rhamnoisorobin to kaempferol was slower than
that of afzelin. The difference in the metabolic rate between afzelin and a-
rhamnoisorobin may due to the location of the rhamnosyl units linked with the parent
nucleus. The predicted metabolic pathway of grosvenorine in the intestinal bacteria is
shown in the Scheme.
4. Anticomplement Activity. The invertebrate complement system can be activated
by a cascade mechanism through either the classical pathway (CP) or the alternative
pathway (AP) [21]. In the present study, complement hemolysis assays were used to
evaluate the anticomplement activities of all of the samples.

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Fig. 2. The time course of the metabolism of grosvenorine by human intestinal bacteria. The values of
the peak areas were provided by HPLC automatic integration. Chromatographic condition was
presented in Fig. 1.
4.1. Inhibition of the Classical Pathway of Complement. The anticomplement activity
through the classical pathway was expressed as the CH50 value, which is the concen-
tration that inhibited 50% of sheep erythrocyte hemolysis (Fig. 3). Although gros-
venorine did not show any obvious anticomplement activity (CH50 >500 mg/ml), its four
metabolites all exhibited significant activities. To the best of our knowledge, this is the
first report of the anticomplement activities of kaempferitrin, afzelin, and a-rham-
noisorobin. The metabolite kaempferol possessed the most potent activity in inhibiting
activation through the classic pathway, with a CH50 value of 17.0Æ5.2 mg/ml. Our
research indicated that the inhibitory potencies against the complement system were
enhanced with the increased number of free OH groups on the A-ring of 5,7-dihydroxy-
flavone, and the presence of the 3-OH group may also enhance the anticomplement
activity of flavonols. This result was consistent with previously published reports [22].
4.2. Inhibition of the Alternative Pathway of Complement. The anticomplement
activity through the alternative pathway was expressed as the AP50 value, which is the
concentration that inhibited 50% of rabbit erythrocyte hemolysis (Fig. 3). Afzelin, a-
rhamnoisorobin, and kaempferol blocked the hemolysis of rabbit erythrocytes in a
dose-dependent manner. The AP50 values were 125.5Æ27.1 mg/ml for a-rhamnoisor-
obin, 172.0Æ21.2 mg/ml for afzelin, and 29.1Æ7.4 mg/ml for kaempferol. Chu reported
that the glycosylation of 3-OH and 7-OH could affect the anticomplement activity
of flavonols [23]. In the present study, the metabolites of grosvenorine were found
to be more active than grosvenorine, indicating that glycosylation could decrease the
activity of kaempferol. Interestingly, a-rhamnoisorobin was more effective on the anti-
complement activity than afzelin, further indicating that the free OH position also
significantly affected the anticomplement activities of flavonols.

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Scheme. The Possible Metabolic Pathway of Grosvenorine in Human Intestinal Bacteria
5. Antioxidant Activities. 5.1. ABTS and DPPH Radical Scavenging Activities. The
ABTS and DPPH radical scavenging activities of the sample were expressed as EC50
values (the effective concentration that resulted in 50% of scavenged radicals). The
EC50 values of grosvenorine and its four metabolites were found to vary from 7.7 to

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
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Fig. 3. Anticomplement activity of grosvenorine and its metabolites. CH50: concentrations that resulted in
50% inhibition of sheep erythrocyte hemolysis; AP50: concentrations that resulted in 50% inhibition of
rabbit erythrocyte hemolysis. Heparin was used as a positive control. The data are presented as the
meanÆSD (n ¼ 3).
87.8 mg/ml in the ABTS assay and from 9.1 to 79.4 mg/ml in the DPPH assay, indicating
that grosvenorine and its metabolites possessed radical-scavenging activities. It has
been reported that the degree of hydroxylation and the positions of the OH groups in
flavonoids could result in increased antioxidant activity, as it can either confer higher
stability to the aroxyl radical by electron delocalization or act as the preferred binding
site for trace metals [24][25]. The results in the present study were consistent with the
literature, and additional evidence revealed that the flavonoid glycoside could be
transformed to a secondary glycoside and an aglycon (which has stronger antioxidant
activities) by human intestinal bacteria (Fig. 4).
5.2. Superoxide Anion Radical Scavenging Activity. The assay for determining
superoxide anion radical scavenging activity is based on the xanthine/xanthine oxidase
reaction system. In this reaction system, the generation of superoxide anion radicals
simulates the xanthine/xanthine oxidase reaction system. Upon electron transfer in the
presence of Gressꢁs reagent, the reaction system exhibits a purple color. The scavenging
activity values for the tested compounds, at a concentration of 50 mg/ml, were varied
from 19.3% to 67.6%. Among them, kaempferol was the strongest scavenger (Fig. 5). It
is reported that the flavonoids with more OH groups could provide more H-atoms for
the conjugation with free radicals, thus providing stronger antioxidant activities [26].
Our results also demonstrated that the deglycosylation of flavonoid glycosides
could enhance their antioxidant activities. In the present study, the antioxidant activity
of a-rhamnoisorobin was stronger than that of afzelin. It could be explained that
the HOÀC(3) has a lower dissociation energy than that of HOÀC(5) and is easy to
dissociate. Thus, a molecule with 3-OH possesses a stronger antioxidant activity
compared to one with 5-OH [27].
6. Antibacterial Activities. The primary antibacterial screening was carried out using
the agar disc-diffusion method. The five flavonoids tested were all observed to be

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Fig. 4. The ABTS and DPPH radical scavenging activities of grosvenorine and its metabolites. EC50
values (the effective concentration resulted in 50% radicals were scavenged) were used to express the
radical scavenging activity. Trolox was used as a positive control. The data are presented as the meanÆ
SD (n ¼ 3).
Fig. 5. The superoxide anion radical scavenging activity of grosvenorine and its metabolites. The initial
sample concentration was 50 mg/ml. Trolox was used as a positive control. The data are presented as the
meanÆSD (n ¼ 3).
active against Gram-positive bacteria, but not against Gram-negative bacteria (Table).
For the first time, grosvenorine, kaempferitrin, and a-rhamnoisorobin were shown to
inhibit both MRSA and MSSA.

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Table. The Antibacterial Activity of Grosvenorine and its Metabolites
Inhibition zone diametera) [mm] (meanÆSD, n¼3)
Sample
MRSA
MSSA
Entero-
E. coli
P. aeuro-
K. pneu-
coccus
ginosa
moniae
Grosvenorine
Kaempferitrin
Afzelin
7.0Æ0.8
10.1Æ0.4
11.8Æ0.9
8.1Æ0.6
12.2Æ0.5
13.1Æ0.7
14.2Æ0.6
16.1Æ1.1
4.1Æ0.5
5.9Æ0.7
8.1Æ0.6
10.2Æ0.4
13.9Æ0.8
–b)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
a-Rhamnoisorobin 13.1Æ0.8
Kaempferol
16.2Æ0.7
Positive control
24.8Æ1.2c) 23.9Æ0.7c) 20.8Æ0.4c) 19.5Æ0.6d) 27.4Æ0.9d) 22.6Æ0.5d)
Negative controle)
–
–
–
–
–
–
a) Sample concentration: 250 mg/ml in 5% (v/v) DMSO solution. b) Not active. c) Ampicillin. d) Levo-
floxacin. e) 5% (v/v) DMSO solution.
To further evaluate the antibacterial activity of the samples, the minimal inhibitory
concentration (MIC) of each compound was determined (Fig. 6). The MIC values of
grosvenorine and its four metabolites against Gram-positive bacteria were all less than
70 mg/ml, indicating that the grosvenorine and its four metabolites exhibited
antibacterial activity. Kaempferol had the best inhibitory activity, of which MSSA
was the most sensitive microorganism. In addition to the Enterococcus strain, a
common conditioned pathogen, afzelin, and a-rhamnoisorobin were found to possess
significant inhibitory activity against both MSSA and MRSA as well. It is known that
the flavonols are more hydrophobic and can penetrate the bacterial phospholipid
membrane more easily to exert their intracellular inhibitory activity compared to their
glycosides [28]. Our results indicated that flavonoids with fewer sugar units
demonstrated better antibacterial activity.
Fig. 6. The MIC values [mg/ml] of grosvenorine and its metabolites
Conclusions. – In this study, for the first time, grosvenorine from the fruits of S.
grosvenorii (Swingle) C. Jeffrey was observed to possess substantial antibacterial
and antioxidant activities. Interestingly, grosvenorine could be transformed by the

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human intestinal bacteria to kaempferitrin, afzelin, a-rhamnoisorobin, and kaempferol,
which all possess the stronger antibacterial, antioxidant, and anticomplement activities.
Grosvenorine could provide natural antibacterial, antioxidant, and anti-complement
benefits to promote human health and prevent related diseases. These results also
indicated that the presence of intestinal bacteria is believed to play an important role in
the gastrointestinal metabolism of grosvenorine and significantly influence its activities.
This study was financially supported by the National Natural Sciences Foundation of the Peopleꢀs
Republic of China (No. 81173551; No. 81374067) and the Shanghai Key Laboratory of Psychotic
Disorders (13dz2260500). The authors would like to thank Dr. Bona Dai at the Instrumental Analysis
Center of Shanghai Jiao Tong University for the NMR measurements and Dr. Changming Mo at the
Guangxi Medicinal Botanical Garden for the sample photograph.
Experimental Part
Plant Material. The dried fruits of S. grosvenorii were collected in December 2010 from Guilin,
Guangxi Province, P. R. China, and authenticated by one of the authors (Mengyue Wang). A voucher
specimen (GX20101209) has been deposited with the herbarium at the School of Pharmacy, Shanghai
Jiao Tong University, Shanghai, P. R. China.
Chemicals and Reagents. Sheep erythrocytes, rabbit erythrocytes, and anti-sheep erythrocyte
antibody were purchased from Shanghai Fortune Biological Technological Co., Ltd. (Shanghai, P. R.
China). Human serum was obtained from healthy adult donors. Guinea pig serum was obtained from
healthy guinea pigs from the Laboratory Animal Research Center of Fudan University. Heparin (sodium
salt, 160 IU/mg) and HZ-801 macroporous resin were purchased from China National Medicines Co. Ltd.
(Shanghai, P. R. China). 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 2,2’-azinobis(3-ethylbenzo-
thiazoline-6-sulfonic acid) diammonium salt (ABTS), and 6-hydroxy-2,5,7,8-tetramethylchromancar-
boxylic acid (Trolox) were purchased from SigmaÀAldrich Chemical Co. (St. Louis, MO, USA). A
commercial kit (A002) used to determine the superoxide radical-scavenging activity was purchased from
Jiancheng Institute of Biotechnology (Nanjing, P. R. China). Deionized H2O was obtained with a Milli-Q
Water purification system (Millipore, MA, USA).
Strains. Methicillin resistant Staphylococcus aureus (MRSA, ATCC25923), methicillin sensitive
Staphylococcus aureus (MSSA, ATCC25923), Enterococcus (ATCC29212), Escherichia coli
(ATCC8739), Pseudomonas aeuroginosa (ATCC27853), and Klebsiella pneumoniae (ATCC700603)
were provided by Dr. Xiuping Qian, School of Pharmacy, Shanghai Jiao Tong University.
Equipment. Analytical chromatography was performed on an Agilent 1200 liquid chromatography
system, which was equipped with a quaternary solvent delivery system, an auto-sampler, a DAD detector
and an Athena C18-WP column (4.6Â250 mm, 5 mm). Semi-prep. chromatography was conducted using
the Shimadzu LC 2010 AHT liquid chromatography system, which was equipped with a quaternary
solvent delivery system, an auto-sampler, a UV/VIS detector and an Athena C18-WP column (10.0Â
250 mm, 5 mm). Anaerobic incubation was performed in an anaerobic chamber (855-ACB, Plas-Labs
Inc., USA). Optical absorbance was measured using an xMark Microplate Absorbance Spectropho-
tometer (Bio-Rad Co. Ltd., USA) with a spectral range from 200 to 1000 nm and 1 nm step size. The
NMR spectra were acquired using Bruker Avance III 600 spectrometers with a 5 mm TCI CryoProbe.
Isolation and Identification of Grosvenorine. The dried and powered fruits (500 g) were decocted
with 3000 ml H2O for 0.5 h. The suspension was passed through an HZ-801 macroporous resin column
(6.5Â80 cm) at a flow rate of 5 ml/min. The column was first eluted with 50% EtOH followed by 70%
EtOH. The 70% EtOH extract was completely evaporated under reduced pressure to afford a residue.
The residue was chromatographed over a Sephadex LH-20 column (4.0Â100 cm) and eluted first with
60% MeOH followed by 80% MeOH. The 80% MeOH extracts were combined and rechromatographed
over another Sephadex LH-20 column (2.5Â60 cm) and eluted with 80% MeOH again to afford the
1
desired compound. The structure was confirmed by the NMR data. H-NMR ((D6)DMSO, 600 MHz):

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12.62 (s, HOÀC(5)); 10.26 (s, HOÀC(4’)); 7.81 (d, J¼8.6, HÀC(2’,6’)); 6.93 (d, J¼8.6, HÀC(3’,5’)); 6.81
(d, J¼1.7, HÀC(8)); 6.48 (d, J¼1.7, HÀC(6)); 3-rha: 5.30 (d, J¼2.0, HÀC(1’’)); 3.99 (br. s, HÀC(2’’));
3.47–3.49 (m, HÀC(3’’)); 3.12–3.14 (m, HÀC(4’’)); 3.14–3.16 (m, HÀC(5’’)); 0.81 (d, J¼5.2, Me(6’’)); 7-
rha: 5.90 (d, J¼2.0, HÀC(1’’’)); 3.94 (d, J¼2.6, HÀC(2’’’)); 3.68–3.70 (m, HÀC(3’’’)); 3.26–3.31 (m,
HÀC(4’’’)); 3.41–3.44 (m, HÀC(5’’’)); 1.15 (d, J¼6.1, Me(6’’’)); 7-glu: 4.39 (d, J¼7.8, HÀC(1’’’’)); 3.05–
3.07 (m, HÀC(2’’’’)); 3.16–3.18 (m, HÀC(3’’’’)); 3.08–3.09 (m, HÀC(4’’’’)); 3.16–3.18 (m, HÀC(5’’’’));
3.48–3.51 (m, HaÀC(6’’’’)); 3.65–3.67 (m, HbÀC(6’’’’)). 13C-NMR ((D6)DMSO, 150 MHz): 157.7 (C(2));
134.4 (C(3)); 177.8 (C(4)); 160.8 (C(5)); 99.4 (C(6)); 161.3 (C(7)); 94.5 (C(8)); 156.0 (C(9)); 105.7
(C(10)); 120.2 (C(1’)); 130.6 (C(2’,6’)); 115.3 (C(3’,5’)); 160.1 (C(4’)); 3-rha: 101.8 (C(1’’)); 70.0 (C(2’’));
70.2 (C(3’’)); 71.0 (C(4’’)); 70.6 (C(5’’)); 17.4 (C(6’’)); 7-rha: 97.1 (C(1’’’)); 79.8 (C(2’’’)); 70.0 (C(3’’’)); 72.0
(C(4’’’)); 69.8 (C(5’’’)); 17.7 (C(6’’’)); 7-glu: 105.6 (C(1’’’’)); 73.8 (C(2’’’’)); 76.6 (C(3’’’’)); 69.8 (C(4’’’’));
76.1 (C(5’’’’)); 60.9 (C(6’’’’)).
Incubation of Grosvenorine with Artificial Gastric Juice and Intestinal Juice. Two mg of grosvenorine
were added to 10 ml of artificial gastric juice (0.08m HCl containing 100 mg pepsin, pH¼1.5) and
artificial intestinal juice (0.05m KH2PO4 containing 100 mg trypsin, pH¼6.8), resp. [29]. The mixture was
incubated at 378 for 24 h; then, 1 ml of incubated soln. was transferred to a 10-ml volumetric centrifuge
tube at 1, 3, 6, 12, and 24 h separately and adjusted to a pH of 7.0 with 0.1 mol/l NaOH. A four-fold
volume of MeOH was added to the incubated soln. immediately and mixed. The mixture was centrifuged
at 4800 rpm for 20 min, and then, the supernatant was evaporated under reduced pressure (458) to afford
a residue. The residue was dissolved in 5 ml of MeOH, filtered through a 0.22 mm membrane and then
analyzed by HPLC.
Incubation of Grosvenorine with Human Intestinal Bacteria. Fresh fecal samples obtained from eight
healthy volunteers were homogenized with 25 volumes of GAM broth. The sediments were removed by
filtration through three pieces of gauze to afford a human intestinal bacterial suspension. Subsequently,
4 mg of grosvenorine were added to 150 ml of the bacterial suspension, and the mixture was incubated at
378 for 24 h in an 855-ACB anaerobic chamber, where the air had been replaced with a gas mixture (H2
5%, CO2 10%, N2 85%). Five ml of incubated soln. were transferred to a 50-ml volumetric centrifuge
tube at 1, 3, 6, 12, and 24 h. A four-fold volume of H2O-sat. AcOEt was added to the incubated soln.
immediately and mixed. The mixture was centrifuged at 4800 rpm for 20 min, and then the supernatant
was evaporated under reduced pressure (458) to give a residue. The residue was dissolved in 3 ml of
MeOH, filtered through a 0.22 mm membrane, and analyzed by HPLC.
HPLC Analysis. HPLC Analysis was performed on an Agilent 1200 system. The injection volume
was 10 ml. The eluent was 0.1% HCOOH soln. in MeCN. A linear gradient solvent system of 10% MeCN
to 100% MeCN was used over a 40-min period at a flow rate of 1.0 ml/min. The detection wavelength was
set at 280 nm, and the column was thermostated at 308.
Isolation and Identification of Grosvenorineꢀs Metabolites. Three hundred mg of grosvenorine were
added to 1200 ml of human fecal suspension, and the mixture was incubated at 378 in an anaerobic
incubator for 6 h. The cultured mixture was removed and extracted with H2O-sat. AcOEt (400 ml) twice.
The extract was evaporated under reduced pressure to afford a residue, which was then dissolved in
MeOH and separated using a Shimadzu LC 2010 AHT liquid chromatography system to afford the pure
metabolites.
Kaempferitrin. 1H-NMR ((D6)DMSO, 600 MHz): 12.61 (s, HOÀC(5)); 10.25 (s, HOÀC(4’)); 7.80 (d,
J¼8.6, HÀC(2’,6’)); 6.92 (d, J¼8.6, HÀC(3’,5’)); 6.79 (d, J¼1.7, HÀC(8)); 6.46 (d, J¼1.7, HÀC(6)); 3-
rha: 5.30 (d, J¼2.0, HÀC(1’’)); 3.99 (br. s, HÀC(2’’)); 3.47–3.50 (m, HÀC(3’’)); 3.13–3.16 (m, HÀC(4’’));
3.11–3.13 (m, HÀC(5’’)); 0.81 (d, J¼5.5, Me(6’’)); 7-rha: 5.55 (d, J¼2.0, HÀC(1’’’)); 3.84 (br. s,
HÀC(2’’’)); 3.62–3.65 (m, HÀC(3’’’)); 3.28–3.31 (m, HÀC(4’’’)); 3.41–3.46 (m, HÀC(5’’’)); 1.13 (d, J¼6.1,
Me(6’’’)). 13C-NMR ((D6)DMSO, 150 MHz): 157.7 (C(2)); 134.4 (C(3)); 177.8 (C(4)); 160.8 (C(5)); 99.4
(C(6)); 161.6 (C(7)); 94.5 (C(8)); 156.0 (C(9)); 105.7 (C(10)); 120.2 (C(1’)); 130.6 (C(2’,6’)); 115.3
(C(3’,5’)); 160.1 (C(4’)); 3-rha: 101.8 (C(1’’)); 70.0 (C(2’’)); 70.2 (C(3’’)); 71.0 (C(4’’)); 70.6 (C(5’’)); 17.4
(C(6’’)); 7-rha: 98.3 (C(1’’’)); 69.7 (C(2’’’)); 70.1 (C(3’’’)); 71.5 (C(4’’’)); 70.0 (C(5’’’)); 17.8 (C(6’’’)).
Anticomplement Activity Evaluation of Grosvenorine and its Metabolites. 1. Anticomplement Activity
through the Classical Pathway (CP). Based on Leeꢁs method [30], sensitized erythrocytes (EA) were
prepared by incubating sheep erythrocytes with rabbit anti-sheep erythrocyte antibodies in GVB2þ

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(Veronal buffered saline containing 0.1% gelatin, 0.5 mm Mg2þ, and 0.15 mm Ca2þ ). The samples, as well
as heparin (which was used as a reference), were dissolved in GVB2þ. Guinea pig serum was used as the
complement source. The 1:40 diluted guinea pig serum was chosen to elicit submaximal lysis in the
absence of complement inhibitors. The mixture was centrifuged immediately. The optical density of the
supernatant was measured at 405 nm with a spectrophotometer. The sample control absorbance was
subtracted from each value to obtain the corrected absorbance, and the percent inhibition was calculated.
The anticomplement activity through the classical pathway was expressed as a CH50 value.
2. Anticomplement Activity through the Alternative Pathway (AP). Based on Klerxꢁs method [31],
various dilutions of each sample were prepared in GVB-Mg-EGTA (Veronal buffered saline containing
0.1% gelatin, 2.5 mm Mg2þ, and 8 mm EGTA). After pre-incubation of the dilutions of each sample
(150 ml) with 1:10 diluted normal human serum (150 ml) in an enzyme-labeled plate at 378 for 10 min,
200 ml of rabbit erythrocytes were added. Following a second incubation step at 378 for 30 min, cell lysis
was determined as described above. Controls for all of the banks, 100% lysis and a sample control were
included. The anticomplement activity through the alternative pathway was expressed as an AP50 value.
Antioxidant Activities Determination of Grosvenorine and its Metabolites. 1. ABTS Radical-
Scavenging Activity Assay. The ABTS radical scavenging activity assay was carried out via ABTS cation
radical decolorization with minor modifications [32]. For each sample, different concentrations were
prepared with MeOH. The ABTS cation radical was prepared by reacting 15 ml of 7 mmol/l ABTS H2O
soln. with 264 ml of 140 mmo/l K2S2O8. The mixture was incubated in the dark at r.t. for 16 h before use.
Prior to assaying, the ABTS working reagent was diluted with MeOH to give an absorbance of 0.50Æ0.02
at 734 nm and equilibrated at r.t. The mixtures in the 96-well plates consisted of the sample (50 ml) and
the ABTS MeOH working soln. (100 ml). The mixture was stirred and left to stand for 10 min in the dark,
after which the absorbance was taken at 734 nm. All measurements were performed in triplicate. The
percentage scavenging effect was calculated as follows: Scavenging rate¼[1À(A1 ÀA2)/A0]Â100%,
where A0 is the absorbance of the blank control (without sample), A1 is the absorbance in the presence of
the sample, and A2 is the absorbance of sample without ABTS working soln. The scavenging ability of the
samples was expressed as an EC50 value.
2. DPPH Radical-Scavenging Activity Assay. H-Donating activity was measured by direct H-
donation to the DPPH radical as previously reported with minor modifications [33]. Samples were
prepared in the same manner as the ABTS assay. The mixtures in the 96-well plates consisted of 100 ml of
sample and 100 ml of DPPH radical (0.2 mmol/l) dissolved in MeOH. The mixture was stirred and left to
stand for 15 min in the dark, after which the absorbance was measured at 517 nm. All measurements were
performed in triplicate. The percentage scavenging effect was calculated as follows: Scavenging rate¼
[1À(A1 ÀA2)/A0]Â100%, where A0 is the absorbance of the blank control (without sample), A1 is the
absorbance in the presence of the sample, and A2 is the absorbance of sample without the DPPH radical.
The scavenging ability of the samples was expressed as an EC50 value.
3. Superoxide Radical-Scavenging Activity. A commercially available kit was used for the superoxide
radical-scavenging activity assay. The superoxide radicals were generated by the xanthine/xanthine
oxidase system and reacted with 2,4-iodiphenyl-3,4-nitrophenyl-5-phenyltetrazolium chloride to form
formazan, a colored compound that can be spectrophotometrically quantified at 550 nm [34]. The
production of formazan is inversely related to the superoxide radical-scavenging activity of the samples
tested. The final results were expressed as the degree of inhibition of formazan production. The samples
were prepared in the same manner as for the ABTS assay. An adjusted concentration in the assay of
50 mg/ml was used. Testing was in accordance with the manufacturerꢁs instructions. All measurements
were performed in triplicate. The percentage scavenging effect was calculated as follows: Scavenging
activity¼(Acontrol ÀAsample)/Acontrol Â100%, where Acontrol and Asample are the average absorbance of the
MeOH control and the sample, resp.
Antibacterial Activity Evaluation of Grosvenorine and its Metabolites. 1. Determination of the
Antibacterial Activity by the Agar Disc-Diffusion Method. The antibacterial effect of all of the samples
was determined in Petri dishes with 20 ml of nutrient agar plus 0.2 ml of microorganism suspension
(108 CFU/ml, OD 0.1, l¼590 nm). Samples and positive controls were dissolved in 5% DMSO soln. at a
concentration of 250 mg/ml. Once the agar had solidified, 200 ml of each sample and 5% DMSO soln.
(negative control) was added to wells 3 mm in diameter. The plates were incubated at 378 for 24 h, and

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1663
then the inhibited halos [mm] were determined. The experiment was repeated in triplicate, and the
antibacterial effect was evaluated by the measure of inhibited halos that had formed around the wells
[35].
2. Determination of Minimal Inhibitory Concentration (MIC). Samples and positive controls were
dissolved in a 5% DMSO soln. at an initial concentration of 2500 mg/ml. Two-fold serial dilutions of the
soln. were then prepared (2500, 1250, ..., and 19.5 mg/ml). Inoculated nutrient broth with 1% text
bacteria strains (108 cuf/ml, OD 0.1, l¼590 nm) was prepared. Eight concentrations of each sample using
three test bacterial strains were carried out in triplicate in sterile round-bottom 96-well plates to compare
the samples with the non-inoculated nutrient broth. In each well, 0.1 ml of the sample was added to 0.9 ml
of inoculated nutrient broth to give final concentrations of 250, 125, ..., and 1.95 mg/ml. Plates were
incubated at 378 for 24 h. The MIC values were determined as the lowest concentration that completely
inhibited visible growth of the bacterial as detected by the unaided eye [11].
Supplementary Material. The 1H-, 13C-, and 2D-NMR (1H,1H-COSY, HSQC, and HMBC) spectra of
grosvenorine and kaempferitrin are available in the supplementary material and can be obtained from
the authors.
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Received October 28, 2014