Bioorganic & Medicinal Chemistry 25 (2017) 28782882

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Identification of spirobisnaphthalene derivatives with anti-tumor

activities from the endophytic fungus Rhytidhysteron rufulum AS21B
Ittipon Siridechakorn a, Zongwei Yue a, Yanisa Mittraphab b, Xiaoguang Lei a, Khanitha Pudhom b,
a
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical
Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing
100871, China
b
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: The cultivation of the mangrove-derived fungus Rhytidhysteron rufulum AS21B in acidic condition chan-
Received 28 January 2017 ged its secondary metabolite profile. Investigation of the culture broth extract led to the isolation and
Revised 23 February 2017 identification of two new spirobisnaphthalenes (1 and 2) together with eleven known compounds (3
Accepted 24 February 2017
13) from the crude extract of the fungus grown under an acidic condition as well as six known com-
Available online 27 February 2017
pounds (4, 10, 1417) were isolated from the crude extract of the fungus grown under a neutral condi-
tion. Their structures were elucidated on the basis of extensive spectroscopic data. The isolated
Keywords:
compounds were evaluated for their cytotoxicity against two human cancer cell lines, Ramos lymphoma
Endophytic fungi
Rhytidhysteron rufulum
and drug resistant NSCLC H1975. Compounds 2 and 10 displayed the most promising anti-tumor activity
Spirobisnaphthalene against both cancer cell lines.
Anti-cancer activity 2017 Elsevier Ltd. All rights reserved.
Fungal cultivation

1. Introduction As a result, alteration of culture media conditions may be of

Endophytic fungi are a rich source of secondary metabolites,
exhibiting a wide range of pharmacological activities.13 Plant-
derived fungi from mangrove forests have received much attention
from pharmaceutical and natural product chemists are interested
in this unique ecosystem.46 Spirobisnaphthalenes are a group of
compounds consisting of two naphthalene-derived C10 units
linked together through a spiroketal linkage. Examples of spirobis-
naphthalenes include the palmarumycins,7,8 decaspirones,7,8
preussomerins9,10 and spiro-nonadiene,11 which have been mainly
isolated from fungi. They have been reported to possess a variety of
biological activities such as antibacterial,12 antifungal,12 anti-
cancer,13 and antileishmanial10 activities.
Endophytic fungi have been able to adapt themselves to reside
in the living tissues of virtually all plants. The metabolomes of
endophytic fungi thus display great diversity and adaptability.14,15
Moreover, varying the conditions of the fungal cultivation, such as
pH or addition of epigenetic modifiers, had led to the production of
higher amounts of some components or even to the production of
some new metabolites.1618
Corresponding author.
E-mail address: khanitha.p@chula.ac.th (K. Pudhom).
great value in the discovery of new compounds. In the course of
our ongoing search for new bioactive compounds from man-
grove-derived fungi, chemical investigations of the Rhytidhysteron
rufulum AS21B fungus under acidic (pH
5) and normal cultivation
conditions were performed. This led to the isolation and character-
ization of two new spirobisnaphthalenes, rhytidenones G and H (1
and 2), together with fifteen known compounds. Isolated com-
pounds were further evaluated for their anti-tumor activities
against two human cancer cell lines, Ramos and H1975.
2. Results and discussion
The EtOAc extracts of the R. rufulum AS21B grown in normal and
acidic SDB broth were successively purified by column chromatogra-
phy. Cultivation in acidic condition led to the isolation of two novel
spirobisnaphthalenes, rhytidenones G and H (1 and 2), along with ele-
ven known compounds: deoxypreussomerin B (3),19 palmarumycin
CP17 (4),10 1-oxo-1,4-dihydronapthalene-4-spiro-20 -naptho[400 -
hydroxy-100 ,800 -de][10 ,30 ]-dioxine (5),20 preussomerin EG4 (6),21
CJ-12,371 (7),22 4-O-methyl-CJ-12,371 (8),22 rhytidenone E (9),23
rhytidenone F (10),23 palmarumycin C5 (11),24 rhytidone A (12)25
and 4,8-dihydroxy-3,4-dihydronaphthalen-1(2H)-one (13),26 while
culture in normal condition (pH
7) gave six known compounds

http://dx.doi.org/10.1016/j.bmc.2017.02.054
0968-0896/ 2017 Elsevier Ltd. All rights reserved.
 I. Siridechakorn et al. / Bioorganic & Medicinal Chemistry 25 (2017) 28782882 2879

Rhytidenone G (1), a24 D 92.8 (c 0.10, MeOH), was obtained as a

yellow viscous oil. Its molecular formula, C20H16O5, was deduced
by HR-ESI-MS which showed the molecular ion peak at m/z
359.0941 [M+Na]+ (calcd. for C20H16O5Na, 359.0895). The IR spec-
trum showed the hydroxyl functionality at 3389 cm1. The combi-
nation of 1H, 13C, and HSQC NMR data for 1 (Table 1) displayed the
typical signals expected for a 1,8-disubstituted naphthalene, due to
the HMBC correlations between H-30 (7.48, dd, J = 8.0, 1.2 Hz) and
C-10 (147.8); C-4a0 (135.2), H-40 (7.57, d, J = 8.0 Hz) and C-20
(110.7); C-50 (121.4); C-8a0 (114.6), H-60 (7.48, dd, J = 8.0, 1.2 Hz)
and C-4a0 (135.2); C-8 (148.2), H-70 (6.96, d, J = 8.0 Hz) and C-50
(121.4); C-8a0 (114.6), as well as COSY correlations of H-20 /H-30 ,
H-30 /H-40 , H-50 /H-60 , and H-60 /H-70 (Fig. 2). Furthermore, the pres-
ence of a naphthalene subunit with an epoxide moiety [dH 3.41 (d,
J = 4.0 Hz), H-6 and 3.77 (t, J = 4.0 Hz), H-7] that conjugated with a,
b-unsaturated ketone at C-5 was established by HMBC correlation
between H-6 and C-4a (131.3); C-5 (195.2), H-7 and C-5 (192.5); C-
8a (45.1) together with COSY correlations of 1 was interpreted the
existence of the spin systems corresponding to the C-2 C-4 and C-8
C-8a. According to our previous isolation of compounds from this
fungal strain,23 compound 1 was recognized as a spirobisnaph-
thalene derivative with a spiroketal-linked 1,8-dioxynaphthalene
unit via a spiroketal bridge, confirming by HMBC correlation of
H-2 (2.19, 1.90, m) with C-1 (103.3). Moreover, the relative config-
uration of 1 was determined by NOESY correlations of H-8/H-8a
and H-6/H-7.
Rhytidenone H (2), a24
D 57.4 (c 0.15, MeOH) was obtained as a
pale yellow gum. Its molecular formula, C22H18O5, was established
by HR-ESI-MS which showed the molecular ion peak at m/z
385.1186 [M+Na]+ (calcd. for C22H18O5Na, 385.1152). The NMR
data of 2 (Table 1) were similar to those of 1, except for the upfield
shift of carbonyl ketone at C-5 to dC 187.7. This finding was con-
firmed by HMBC correlations between H-6 (6.17, d, J = 10.0 Hz)
and C-4a (131.5); C-8 (63.9), H-7 (6.91, d, J = 10.0 Hz) and C-5
(187.7); C-8a (45.1) (Fig. 2). Furthermore, the presence of an acetyl
group [dH 2.11 (s), dC 21.0 and 169.9] instead of hydroxyl at C-8
established a downfield shift of H-8 (dH 5.69, dd, J = 5.6, 3.2 Hz)
on the basis of the mesomeric effect of the acetyl group. Moreover,
the relative configuration of 2 was determined by NOESY correla-
tion of H-8 with H-8a.
The anti-tumor activities of compounds 113 were evaluated
against Ramos and H1975 cell lines (Table 2). Ramos and H1975 cell
lines were derived from a human Burkitts lymphoma and non-small
cell lung cancer tissue respectively, and is hence representative of
this type of neoplasm. Though Burkitts lymphoma (BL) is one of
the most curable hematological malignancies, this disease is also
one of the most aggressive types of cancer, which always occur in
adolescents and young adults even as well as in childhood
patients.28 Ibrutinib (PCI-32765), a Brutons tyrosine kinase inhibi-
tor, was developed by Pharmacyclics partnered with Johnson &
Johnson and was granted an accelerated approval by US FDA in
November 2013, for the treatment B cell cancers like mantle cell
lymphoma. Lung cancer is one of the most frequently diagnosed
types of cancer among males and females in China and in Western
Fig. 1. Compounds isolated from R. rufulum AS21B strain.
countries, and it remains the leading cause of cancer mortality in
the USA, and non-small-cell lung carcinoma (NSCLC) which is char-
acterized by aberrant activity of EGFR accounts for about 85% of all
lung cancers. Though the kinase inhibitors, gefitinib and erlotinib
including compounds 4 and 10, and other four compounds: rhytidone
are effective clinical therapies for this disease, approximately 50%
B (14)25 and C (15),25 MK3018 (16)27 and palmarumycin CR1 (17)20
of patients who initially respond to therapy will relapse due to emer-
(Fig. 1). Interestingly, it was found that the culture in acidic medium
gence of drug resistance H1975 cell line with EGFR mutation L858R/
enhanced the production of compounds 4 and 10, with 4-fold and 8-
T790M.29,30 To overcome this drug resistance, new inhibitors effec-
fold increasing, respectively, which are present in minor quantities
tive for H1975 cell line with less toxic and side effect are urgent to be
under normal culture condition. The structures of the known com-
developed. Afatinib (BIBW 2992), an irreversible, tyrosine kinase
pounds were determined by comparison of their NMR spectroscopic
inhibitor (TKI) of ErbB receptor family members, is effective in the
data with those in the literature.
2880 I. Siridechakorn et al. / Bioorganic & Medicinal Chemistry 25 (2017) 28782882

Table 1
1 13
H (400 MHz) and C (100 MHz) NMR data of compounds 1 and 2.

Position 1a 2b
dC, type dH, mult. (J in Hz) dC, type dH, mult. (J in Hz)
1 103.3 C 101.2 C
2 28.8 CH2 2.19, m1.90, m 29.2 CH2 2.18, m
3 23.5 CH2 2.37, m 22.6 CH2 2.49, m2.59, m
4 139.3 CH 7.10, dd (6.4, 3.6) 136.7 CH 7.08, br m
4a 131.3 C 131.5 C
5 192.5 C 187.7 C
6 55.6 CH 3.41, d (4.0) 132.9 CH 6.17, d (10.0)
7 57.2 CH 3.77, t (4.0) 142.0 CH 6.91, d (10.0)
8 64.3 CH 5.10, br s 63.9 CH 5.69, dd (5.6, 3.2)
8a 43.6 CH 3.27, br s 45.1 CH 3.31, m
10 147.8 C 146.4 C
20 110.7 CH 6.99, d (8.0) 109.7 CH 6.86, d (7.6)
30 128.6 CH 7.48, dd (8.0, 1.2) 127.8 CH 7.39, t (8.0)
40 121.7 CH 7.57, d (8.0) 120.8 CH 7.50, d (8.0)
4a0 135.2 C 134.3 C
50 121.4 CH 7.56, d (8.0) 120.8 CH 7.49, d (8.0)
60 128.5 CH 7.48, dd (8.0, 1.2) 127.6 CH 7.43, t (8.0)
70 110.3 CH 6.96, d (8.0) 109.4 CH 6.88, d (7.6)
80 148.2 C 147.0 C
8a0 114.6 C 113.5 C
8-OAc 169.9 C@O
8-OAc 21.0 CH3 2.11, s
a
Recorded in CDCl3.
b
Recorded in acetone-d6.

FDA. Among the compounds tested, compound 2 displayed the most

promising anti-tumor activity against both Ramos and H1975 cell
lines with IC50 values of 0.018 and 0.252 lM, respectively. The sec-
ond potent one is compound 10, the deacetylated derivative of 2,
and it gave IC50 values of 0.048 and 1.17 lM, respectively. Com-
pound 2 and 10 may be more effective in anti-tumor activity against
Ramos and H1975 than stand drug Ibrutinib and afatinib, with IC50
values of 28.7 and 1.97 lM. When their structure-activity relation-
ship was considered, it could be concluded that the C-4 a,b-unsatu-
rated ketone moiety was crucially required for their potent anti-
tumor activity against Ramos and H1975 cell lines. This could be
seen in the case of compounds 5 and 6, which had the C-2 a,b-unsat-
urated ketone group. Both compounds exhibited only moderate and
weak activity on Ramos, and completely lost activity on H1975 cell
Fig. 2. Selected HMBC and COSY correlations of compounds 1 and 2. line (IC50 > 100 lM). It is worth mentioning that the a,b-unsaturated
carbonyl compounds always have cytotoxic activity, most likely
through the Michael addition reaction with biomolecules.31
Table 2
Anti-tumor activities of compounds isolated from the crude extract of the fungus
grown under an acidic condition. 3. Conclusion
Compound IC50 (mM)
In this study, we studied the effect of pH of the culture medium
Ramos Cell H1975 Cell on Rhytidhysteron rufulum metabolite profile. The culture in acidic
1 17.98 7.3 condition (pH
5) significantly enhanced the production of its sec-
2 0.018 0.252 ondary metabolites, including two new spirobisnaphthalenes (1
3 18 >100
and 2). Interestingly, the acidic culture condition also remarkably
4 33.1 >100
5 15 >100 increased the production of the most two promising metabolites
6 82.9 >100 (2 and 10) against Ramos and drug resistant H1975 human cancer
7 >100 >100 cell lines with very low IC50 values. Based on their structure-activ-
9 0.461 10.24
ity relationship, we found the C-4 a,b-unsaturated ketone moiety
10 0.048 1.17
11 31.7 >100 was required for their potent anti-tumor activity. These findings
12 >100 >100 suggest that compounds 2 and 10 might be promising leads for
13 23.1 50 leukemia and lung cancer treatments. Their mechanism of action
Ibrutinib 28.7 ND will be further studied and reported in due course.
Afatinib ND 1.97

ND: not determined. 4. Material and method

treatment of lung cancer models, including those with EGF receptor 4.1. General experiment procedures
(EGFR) mutations (L185R/T790M) resistant to reversible first-gener-
ation EGFR inhibitor like Gefitinib. It was developed and marketed The UV spectra were recorded on a Biotek Powerwave XS2
by Boehringer Ingelheim, and was first approved in 2013 by US (USA). The IR spectra were recorded with a PerkinElmer FTS FT-
 I. Siridechakorn et al. / Bioorganic & Medicinal Chemistry 25 (2017) 28782882 2881

IR spectrophotometer. Optical rotations were measured on a Perki- tion L (193.0 mg) by CC Sephadex-LH20 with MeOH to give five
nElmer 341 polarimeter. NMR spectra were acquired on a Varian subfractions (LA-LE). Compound 4 (4.1 mg) was derived from sub-
Mercury-400 Plus NMR spectrometer and 400 MHz Bruker FTNMR fraction LD (101.3 mg) by CC with 25% acetone-hexane.
Ultra Shield with TMS as internal standard. HRESI-MS was carried Rhytidenone G (1): yellow viscous oil, a24 92.8 (c 0.10,
D
out on a micrOTOF-Q II ESI mass spectrometer. HPLC was per- MeOH); UV (MeOH) kmax (log e) 250 (2.42), 260 (2.43) nm; IR
formed on Thermo Finnigan (TSP) with preparative column Inertsil (neat) mmax 3389 cm1; 1H and 13C NMR (acetone-d6, 400 MHz),
ODS-3 (GL Science, 5 lm, 20  250 mm). Column chromatography see Table 1; HRESIMS m/z 359.0941 [M+Na]+ (calcd for C20H16O5-
(CC) was carried out on silica gel (Silicycle, SiliaFlash P60 40-63 mm Na, 359.0895).
and SiliaFlash F60 40-63 mm). Sephadex LH-20 was also used for CC. Rhytidenone H (2): pale yellow gum; a24
D 57.4 (c 0.15, MeOH),
Precoated plates of silica gel 60 F254 were used for analytical
UV (MeOH) kmax (log e) 230 (2.44), 250 (2.45), 260 (2.46) nm; 1H
purposes.
and 13C NMR (CDCl3, 400 MHz), see Table 1; HRESIMS m/z
385.1186 [M+Na]+ (calcd for C20H16O5Na, 385.1152).
4.2. Extraction and isolation
4.3. Anti-tumor assay
The fungus R. rufulum AS21B was isolated from leaves of Azima
sarmentosa, collected from the mangrove forest in Samutsakhon
Human lymphoma Ramos cells and NSCLC H1975 cells were
province, Thailand in July 2008. The strain AS21B was grown on
cultured to the exponential growth phase in RPMI 1640 supple-
potato dextrose agar (PDA) plate at room temperature for 7 days.
mented with 10% (v/v) fetal calf serum in a humidified atmosphere
Five pieces (5  5 mm2) of mycelia agar plugs were inoculated into
containing 5% CO2.
1 L Erlenmeyer flasks (100) containing 200 mL of sabouraud dex-
Cytotoxicity against Ramos and H1975 cells was assessed as fol-
trose broth (SDB) in acidic condition (pH
5) as well as the normal
lows: 4  104 cells seeded onto 96-well plates were incubated with
SDB. The cultivation was kept at room temperature under static
compounds at the indicated concentrations at 37 C for 24 h
conditions for 21 days.
(Ramos) or 72 h (H1975). Cell viability was determined using mea-
The filtration was used to separate the mycelia and broth from
surement ATP level in living cells method by CellTiter-Glo kit
each other. The filtrate was extracted with EtOAc 3 times and evap-
according to the manufacturers instructions. Ibrutinib and afatinib
orated under reduced pressure to give an acidic condition crude
were used as reference drug for Ramos and H1975 cell,
extract (5.53 g). The crude extract was subjected to column chro-
respectively.
matography (CC) over silica gel, eluting with a gradient of ethyl
acetate (EtOAc)-hexane (100% hexane to 100% EtOAc), providing
Acknowledgments
eight fractions (AH). Fraction A (235.8 mg) was recrystallized by
hexane to give solid and mother liquor (ML) fraction. The ML
This work was supported by the Thailand Research Fund (TRF)
(221.1 mg) was further purified by CC with 30% CH2Cl2-hexane giv-
(Grant No. BRG5980002) and the National Natural Science Founda-
ing compound 3 (11.4 mg). Fraction B (98.4 mg) was subjected to
tion of China (Grant No. 21561142002).
CC with 50% CH2Cl2-hexane to yield compound 4 (15.8 mg)
together with three subfractions (BA-BC). Compound 6 (2.7 mg)
was purified from subfraction BC (10.0 mg) by CC with 25% A. Supplementary data
EtOAc-hexane. The recrystallization of fraction C (140.7 mg) from
methanol (MeOH) provided compound 5 (2.7 mg) and ML fraction. Supplementary data associated with this article can be found, in
The ML (138.0 mg) was further separated by CC with 50% CH2Cl2- the online version, at http://dx.doi.org/10.1016/j.bmc.2017.02.054.
hexane yielding six subfractions (CA-CF). Compounds 2 (1.9 mg), 8
(1.0 mg) and 9 (3.5 mg) were derived from subfraction CD References
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