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Antimicrobial Triterpenes from Dillenia philippinensis

Authors:
Consolacion Ragasa at De La Salle University
Consolacion Ragasa
Agnes Alimboyoguen at Cavite State University
Agnes Alimboyoguen
Chien-Chang Shen
Chien-Chang Shen
Chien-Chang Shen
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Abstract and Figures

The air-dried leaves of Dillenia philippinensis, commonly known as “katmon” afforded betulinic acid (1) and 3-oxoolean-12-en-30-oic acid (2) by silica gel chromato-graphy. Their structures were elucidated by extensive 1D and 2D NMR spectroscopy. Compounds 1 and 2 exhibited moderate activity against the fungus Candida albicans and slight activity against the bacteria E. coli, P. aeruginosa, S. aureus, and B. subtilis. Compound 2 exhibited slight activity against T. mentagrophytes, while 1 was inactive against this fungus. Both compounds were inactive against A. niger.
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Philipp. Scient. 46: 78–87
© 2009, University of San Carlos
Antimicrobial Triterpenes from
Dillenia philippinensis
Consolacion Y. Ragasa,1* Agnes B. Alimboyoguen,1 and Chien-Chang Shen2
1Chemistry Department and Center for Natural Sciences and Ecological
Research, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines
2National Research Institute of Chinese Medicine,
155-1, Li-Nong St., Sec 2, Taipei 112, Taiwan
ABSTRACT
The air-dried leaves of Dillenia philippinensis,
commonly known as “katmon” afforded betulinic acid (1) and
3-oxoolean-12-en-30-oic acid (2) by silica gel chromato-
graphy. Their structures were elucidated by extensive 1D and
2D NMR spectroscopy. Compounds 1 and 2 exhibited
moderate activity against the fungus Candida albicans and
slight activity against the bacteria E. coli, P. aeruginosa, S.
aureus, and B. subtilis. Compound 2 exhibited slight activity
against T. mentagrophytes, while 1 was inactive against this
fungus. Both compounds were inactive against A. niger.
Key words: Dillenia philippinensis, Dilleniaceae, katmon,
betulinic acid, 3-oxoolean-12-en-30-oic acid,
antimicrobial
INTRODUCTION
Dillenia philippinensis, commonly known as “katmon” is an
endemic and threatened Philippine tree found in forests at low and
medium altitudes. The juice of the fruit is used for the treatment of cough
(Quisumbing 1978). There are no reported chemical studies on D.
philippinensis, however congeners of the tree have been studied for their
*Corresponding author: Tel/Fax: (+632) 5360230
E-mail: ragasac@dlsu.edu.ph
Ragasa et al.: Triterpenes from Dillenia philippinensis 79
chemical constituents and biological activities. Betulinic acid, betulinol,
lupeol, gallic acid and ethyl gallate isolated from D. kerrii inhibited the
proliferation of K562 cells. Gallic acid and ethyl gallate also showed
anti-hypoxia effect (Li et al. 2009). Another study reported the isolation
of dillenic acids D and E, 3-oxoolean-12-en-30-oic acid and betulinic
acid from D. papuana (Nick et al. 1995) which are of relevance to our
present report. Dillenic acids A, B, C, and 3-oxoolean-1,12-dien-30-oic
acid isolated from D. papuana exhibited antibacterial activities against B.
subtilis, E. coli and M. luteus (Nick et al. 1994).
We report here the isolation, structure elucidation, and
antimicrobial test results of betulinic acid (1) and 3-oxoolean-12-en-30-
oic acid (2) from the dichloromethane extract of the leaves of D.
philippinensis. This is the first report on the isolation of these compounds
from D. philippinensis.
COOH
HO
H
H
1
10
6
11
14
18
29
30
20
21
24 23
25 26
27
28
1
HOO C
O
H
H
1
47
10
25 11 26
27
18
28
20
22
30 29
14
2
MATERIALS AND METHODS
General Experimental Procedures
NMR spectra were recorded on a Varian Unity Inova
spectrometer in CDCl3 at 500 MHz for 1H NMR and 125 MHz for 13C
NMR spectra. Column chromatography was performed with silica gel 60
(70–230 mesh). Thin layer chromatography was performed with plastic-
backed plates coated with silica gel F254; plates were visualized by
spraying with vanillin–sulfuric acid, followed by warming.
(4)
(3)
80 The Philippine Scientist, Volume 46 (2009)
Sample Collection
The sample was collected from around Lake Buhi, Camarines
Sur in September 2007. It was identified as Dillenia philippinensis by
Noe Gapas of the Philippine National Museum who also collected the
sample. D. philippinensis (voucher specimen # 135) was deposited at the
Chemistry Department, De La Salle University–Manila.
Isolation
The air-dried leaves of D. philippinensis (1 kg) were ground in
an osterizer, soaked in dichloromethane for three days, then filtered. The
filtrate was concentrated under vacuum to afford a crude extract (55 g)
which was chromatographed in increasing proportions of acetone in
dichloromethane at 10% increment. The 30% acetone in dichloro-
methane fraction was rechromatographed (3x) in diethyl ether:
acetonitrile:dichloromethane (0.5:0.5:9) to afford 2 (10 mg, 0.018%).
The 40% and 50% acetone in dichloromethane fractions were combined
and rechromatographed (5x) in diethyl ether:acetonitrile:dichloro-
methane (1:1:8) to afford 1 (25 mg, 0.045%).
Antimicrobial Test
The microorganisms used were obtained from the University of
the Philippines Culture Collection (UPCC). These were Pseudomonas
aeruginosa (UPCC 1244), Bacillus subtilis (UPCC 1149), Escherichia
coli (UPCC 1195), Staphylococcus aureus (UPCC 1143), Candida
albicans (UPCC 2168), Trichophyton mentagrophytes (UPCC 4193) and
Aspergillus niger (UPCC 3701). The test compound was dissolved in
95% ethanol. The activity index was computed by subtracting the
diameter of the well from the diameter of the clearing zone divided by
the diameter of the well. The antimicrobial assay reported in the
literature was employed (Guevara & Recio 1985).
RESULTS AND DISCUSSION
The dichloromethane extract of the air-dried leaves of D.
philippinensis afforded 1 and 2 by silica gel chromatography. Their
Ragasa et al.: Triterpenes from Dillenia philippinensis 81
structures were elucidated by extensive 1D and 2D NMR spectroscopy as
described below.
The 1H NMR spectrum of 1 (Table 1) gave resonances for
methylene olefinic protons at δ 4.62 and 4.70; an allylic methyl at δ 1.69;
an oxymethine proton at δ 3.20; and five methyl singlets at δ 0.74, 0.80,
0.92, 0.95, and 0.96. The shielded region of the spectrum indicated
overlapping resonances for methylene and methine protons. These
resonances are typical of triterpenes with olefin, hydroxyl, and
carboxylic acid functionalities. Although the carboxylic acid proton was
not detected in the 1H NMR spectrum, 1 contained only six methyl
groups instead of eight expected for a triterpene. Since one of the methyl
groups was converted into an olefin, the remaining methyl must have
been oxidized into a carboxylic acid. This was confirmed by the 13C
NMR spectrum of 1 (Table 1) which gave a resonance for the carboxylic
acid at δ 179.8. An oxymethine carbon (δ 79.0) and olefinic carbons (δ
150.4 and 109.7) were also detected. The remaining twenty-six carbon
resonances were attributed to the methyl, methylene, methine and
quaternary carbons in 1.
The COSY spectrum of 1 gave four isolated spin systems as
follows: H2-1/H2-2/H-3; H-5/H2-6/H2-7; H-9/H2-11/H2-12/H-13/H-18/H-
19/H2-21/H2-22; H2-15/H2-16 (Fig. 1).
1
23
24
21
20
30
29
18
COOH
HO
H
H
4
: HM BC
: CO SY
6
10
15
11
28
Figure 1. 1H–1H COSY and key 1H–13C long-range correlations of 1.
82 The Philippine Scientist, Volume 46 (2009)
Table 1. 500 MHz 1H and 125 MHz 13C NMR spectral data of 1 and 2 in CDCl3.
Position δC, 1 δH mult. (J Hz),* 1 δC, 2 δH mult. (J
Hz),* 2
1 38.7 0.90, 1.69 39.3 1.40, 1.88
2 27.4 1.55, 1.62 34.2 2.35, 2.55
3 79.0 3.20 dd (4.5, 11.5) 217.8 –
4 38.9 – 47.5 –
5 55.3 0.68 55.3 1.32
6 18.3 1.53 (2H) 19.6 1.50, 1.52
7 34.3 1.40 (2H) 32.2 1.39, 1.54
8 40.7 – 39.3 –
9 50.5 1.26 46.9 1.64
10 37.2 – 36.7 –
11 20.8 1.41, 1.43 23.5 1.88, 1.98
12 25.5 1.66, 1.68 122.5 5.30 t (3.5)
13 38.4 2.20 144.3 –
14 42.4 – 41.6 –
15 29.7 1.22, 1.55 26.1 1.02, 1.80
16 32.1 1.42, 2.30 26.9 0.90, 1.95
17 56.3 – 32.0 1.54, 1.38
18 49.3 1.62 48.1 1.98
19 46.9 3.02 dt (10.5, 5.5) 42.6 1.64, 1.86
20 150.4 – 44.0 –
21 30.5 1.41, 1.99 31.1 1.34, 1.92
22 37.0 1.48, 1.96 38.3 1.42, 1.90
23 27.9 0.95 (s) 26.4 1.08 (s)
24 15.3 0.74 (s) 21.5 1.04 (s)
25 16.1 0.80 (s) 15.2 1.05 (s)
26 16.0 0.92 (s) 16.7 1.00 (s)
27 14.7 0.96 (s) 25.9 1.13 (s)
28 179.8 – 28.2 0.80 (s)
29 109.7 4.62 d (2.0), 4.70 d (2.0) 28.7 1.19 (s)
30 19.4 1.69 (s) 182.3 –
*multiplet unless otherwise indicated
Ragasa et al.: Triterpenes from Dillenia philippinensis 83
The 1H and 13C NMR connectivities in 1 (Table 1) were verified
by HMQC. The structure of 1 was elucidated by analysis of the HMBC
2D NMR data with key HMBC correlations shown in Figure 1. Thus, the
hydroxyl was attached to C-3 since long-range correlations were
observed between H2-1, H2-2, H3-23, H3-24 and C-3. The olefin was
assigned to C-20 on the basis of long-range correlations between H3-30
and C-19, C-20, C-29. The carboxylic acid was attributed to C-28 since
long-range correlations were observed between H-18, H2-16, H2-22 and
this carbon. All long-range correlations are consistent with the structure
of 1. Literature search revealed that 1 is betulinic acid. The
confirmatory evidences are the 1H and 13C NMR spectral data of 1 and
betulinic acid (Khaliq et al. 2007) which match in all essential aspects. It
was previously reported as a constituent of D. kerrii (Li et al. 2009) and
D. papuana (Nick et al. 1994). Betulinic acid is a potent anti–HIV agent
(Kashiwada et al. 1996, Hashimoto et al. 1997, Cichewicz & Kouzi
2004) and a cytotoxic agent against malignant brain tumor cells (Fulda et
al. 1999). It also triggers apoptosis in neuroectodermal tumors (Fulda et
al., 1997; Schmidt et al., 1997), induces apoptosis in glioma cells (Wick
et al. 1999), leukemia cells (Ehrhardt et al. 2004) and cancer cells
(Rzeski et al. 2006), and suppresses carcinogen-induced NF-kappa B
activation (Takkada & Aggarwal 2003).
The 1H NMR spectrum of 2 (Table 1) gave resonances for seven
methyl singlets at δ 1.08, 1.04, 1.05, 1.00, 1.13, 0.80, 1.19 and an
olefinic proton at δ 5.30. The 13C NMR spectrum of 2 (Table 1) indicated
thirty carbon resonances, from which the following functionalities were
deduced: olefinic carbons (δ 122.5 and 144.3), carbonyl carbons of a
ketone (δ 217.8), and a carboxylic acid (δ 182.3). These data suggested a
triterpene with olefin, carboxylic acid and ketone functionalities.
The COSY spectrum of 2 gave six isolated spin systems as
follows: H2-1/H2-2; H-5/H2-6/H2-7; H-9/H2-11/H-12; H2-15/H2-16; H-
18/H2-19; H2-21/H2-22 (Fig. 2).
The 1H and 13C NMR connectivities in 2 (Table 1) were verified
by HMQC. The structure of 2 was elucidated by analysis of the HMBC
2D NMR data with key HMBC correlations shown in Figure 2. Thus, the
carbonyl was assigned to C-3 since long-range correlations were
observed between H2-1, H2-2, H3-23, H3-24 and C-3. The olefin was
assigned to C-12 on the basis of long-range correlations between H-9,
H2-11, H-18 and C-12. The carboxylic acid was attributed to C-30 due to
84 The Philippine Scientist, Volume 46 (2009)
long-range correlations between H3-29, H2-19, H2-21 and this carbon. All
long-range correlations are consistent with the structure of 2.
Figure 2. 1H–1H COSY and key 1H–13C long-range correlations of 2.
Literature search revealed that 2 is 3-oxoolean-12-en-30-oic acid
as confirmed by similar 1H and 13C NMR spectral data (Nick et al. 1995).
Compound 2 which was isolated from D. papuana was reported to
exhibit antibacterial activities against B. subtilis, E. coli, and M. luteus
(Nick et al. 1995).
As part of our continuing search for antimicrobial compounds
from Philippine medicinal plants, 1 and 2 were tested for antimicrobial
activity against seven microorganisms (Table 2). Compound 1 at a
concentration of 30 µg indicated antibacterial activity against E. coli with
an activity index (AI) of 0.2, P. aeruginosa (AI = 0.2), S. aureus (AI =
0.1), and B. subtilis (AI = 0.4 thinning), while the standard antibiotic
chloramphenicol gave an AI of 2.8, 1.3, 3.2, and 2.3, respectively. It also
exhibited antifungal activity against C. albicans (AI = 0.2) and T.
mentagrophytes (AI = 0.3), while the standard antibiotic Canesten
indicated an AI of 0.8 and 4.5, respectively. It was inactive against A.
niger. Compound 2 at the same concentration exhibited antibacterial
activity against E. coli (AI = 0.3), P. aeruginosa (AI = 0.3), S. aureus
(AI = 0.4), and B. subtilis (AI = 0.4 thinning). It also exhibited antifungal
activity against C. albicans (AI = 0.3) and T. mentagrophytes (AI = 0.3),
while it was inactive against A. niger.
Ragasa et al.: Triterpenes from Dillenia philippinensis 85
Table 2. Antimicrobial Test Results on 1 and 2.
Organism Sample (30µg) Clearing Zone
(mm)a Activity
Index (AI)
E. coli 1 12 0.2
2
13 0.3
Chloramphenicol
b
23 2.8
P. aeruginosa
1
12 0.2
2 13 0.3
Chloramphenicol
b
14 1.3
S. aureus
1 11 0.1
2 14 0.4
Chloramphenicol
b
25 3.2
B. subtilis
1
(14)
d
(0.4)
2 (14)
d
(0.4)
Chloramphenicol
b
20 2.3
C. albicans 1 12 0.2
2
13 0.3
Canesten, 0.2g
c
18 0.8
T. mentagrophytes
1
13 0.3
2 13 0.3
Canesten, 0.2g
c
55 4.5
A. niger 1 – 0
2
– 0
Canesten, 0.2g
c
23 1.3
a Average of three trials; bChloramphenicol disc – 6 mm diameter;
cContains 1% chlotrimazile; d(–) Thinning of growth, Incomplete inhibition
CONCLUSION
This study afforded two triterpenes betulinic acid (1) and 3-
oxoolean-12-en-30-oic acid (2) from the leaves of Dillenia
philippinensis, which is an endemic and threatened Philippine tree.
Compounds 1 and 2 exhibited antimicrobial properties. Literature search
revealed that 1 is a potent anti–HIV agent and cytotoxic agent against
malignant brain tumor cells. It also triggers apoptosis in neuroectodermal
tumors, glioma cells, leukemia cells and cancer cells, and suppresses
carcinogen-induced NF-kappa B activation. Meanwhile, 2 exhibited
antibacterial activities against B. subtilis, E. coli, and M. luteus. Thus, D.
86 The Philippine Scientist, Volume 46 (2009)
philippinensis, which has no previously reported chemical and biological
activity studies, contains bioactive constituents.
ACKNOWLEDGMENT
The antimicrobial tests were conducted at the University of the
Philippines–Natural Sciences Research Institute (UP–NSRI). Research
grants from the Science Foundation and the University Research
Coordination Office of De La Salle University are gratefully
acknowledged.
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Fulda S, Friesen C, Los M, Scaffide C, Mier W, Benedict M, Nunez G, Debatin
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... Nick et al., (1995) stated that the antibacterial activity of D. papuana, which is in the same genus as D. serrata, is derived from triterpenoid acid compounds. Triterpenoid acid compounds such as Betulinic acid, Dillenic acid A, Dillenic acid B, Dillenic acid C, Dillenic acid D, Dillenic acid E, 3-Oxoolean-1,12-dien-30-oic acid, and 3-Oxoolean-12-en -30-oic acid derived from the plants D. papuana and D. philippinensis is a compound reported to have antibacterial properties (Nick et al., 1994;Nick et al., 1995b;Ragasa et al., 2009). ...
... However, they showed weak inhibition against the fungi Aspergillus fumigatus, A. niger, Candida albicans, C. arriza, C. crusei, Penicillium sp., Rhizopusoryzae, Saccharomyces cerevisiae and Trichoderma viride (Nick et al., 1995a;Wiart et al. al., 2004;Haque et al., 2008;Apu et al., 2010;Smitha et al., 2012). Seven types of triterpenoids from the Dillenia plant have been shown to have antimicrobial action (Nick et al., 1995b;Ragasa et al., 2009). Strong growth inhibition against Escherichia coli, Bacillus subtilis and Micrococcus luteushas been demonstrated by Dillenia papuana (Nick et al., 1995b). ...
... Strong growth inhibition against Escherichia coli, Bacillus subtilis and Micrococcus luteushas been demonstrated by Dillenia papuana (Nick et al., 1995b). The same growth inhibition of Dillenia philippinensis was also reported against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and B. subtilis (Ragasa et al., 2009). These findings suggest that the Dillenia plant has potential as an antimicrobial agent supporting its traditional use for the therapeutic treatment of microbial infections related to diseases such as diarrhea, dysentery, septic chemical infections and skin-related diseases (Sabandar et al., 2016). ...
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... In a study conducted in 2009, silica gel chromatography indicated the presence of betulinic acid and 3-oxoolean-12-en-30-oic acid in D. philippinensis. These compounds exhibited antibacterial and antifungal activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Candida albicans (Ragasa et al., 2009). The most extensively studied species among the genus Dillenia is D. indica. ...
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... The mixture was incubated for another 30 min at 25°C after the addition of 60 µL xanthine solution (150 µM). The reaction was terminated by mixing 25 µL of hydrochloric acid (1 N) solution, and percentage inhibition was calculated utilizing different concentrations of allopurinol (5,10,20,50,100,200, and 300 µg/mL) as the standard. The control solution was prepared similarly, but the addition of hydrochloric acid solution was prior to the addition of xanthine solution (Quy and Xuan, 2019). ...
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Full-text available
  • Dec 2024
The study aimed to provide a taxonomic description of Dillenia sp. and evaluate its antibacterial activity against Escherichia coli (E. coli). The taxonomic description was characterized using the taxonomic keys and field guides. The antibacterial activity of Dillenia sp. was screened using the Kirby-Bauer disk diffusion assay. Taxonomic description confirms Dillenia sp. to be an angiosperm flowering seed plant native to the Philippines, characterized by its round-shaped growth and evergreen foliage. The antibacterial activity of Dillenia sp. bark extracts against E. coli were assessed using different concentrations (100 ppm, 500 ppm, and 1000 ppm) and extraction solvents (ethanolic, aqueous, and decoction). There are significant differences in the antibacterial activity observed among treatments within the decoction extracts, indicating varying effects on antibacterial activity. Post-hoc analysis revealed that concentrations of 100 ppm and 500 ppm were significantly more effective in inhibiting bacterial growth compared to 1000 ppm within the Decoction treatment. Each treatment showed distinct patterns of antibacterial activity, with ethanolic and aqueous extracts displaying relatively consistent activity across different concentrations, while the decoction extract exhibited concentration-dependent antibacterial activity. The study provides evidence of the antibacterial potential of Dillenia sp. bark extracts against Escherichia coli, with implications for further research and potential practical applications in combating bacterial infections. Recommendations include exploring different lower concentration ranges, considering different treatment formulations, investigating combination treatments, and assessing antimicrobial mechanisms to enhance efficacy and guide the development of novel antibacterial strategies.
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... Beyond its common applications, this crop holds significant potential in the medicinal field. Consolacion Ragasa et al. (2009) from De La Salle University concluded that the leaf extract exhibits notable antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, and Trichophyton mentagrophytes. Additionally, it has shown promising anti-cancer properties. ...
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... Interestingly, the fungal endophytes are reported to be good sources of secondary metabolites that can be used to develop novel drugs. However, studies on this plant mainly focus on fruit extract and its medicinal uses, which has been found to have anti-leukemia and antioxidant properties that can remedy coughs and fevers [12,14]. ...
Antibacterial Activities of Fungal Endophytes from Philippine Endemic Plant Dillenia philippinensis: Antibacterial Activities of Fungal Endophytes from Dillenia philippinensis
Article
Full-text available
  • Sep 2023
Fungal endophytes represent a group of microorganisms that establish symbiotic associations with plants and hold significant ecological importance. Their ability to produce a diverse array of biologically active secondary metabolites has garnered considerable interest in the search for novel drug leads. In this study a total of 33 fungal endophytes were isolated from leaf specimens of the Philippine endemic tree Dillenia philippinensis (Rolfe). The morphological characterization of the fungal isolates revealed their taxonomic affiliation with the following eight genera: Alternaria sp., Aspergillus sp., Geotrichum sp., Guignardia sp., Nigrospora sp., Paecilomyces sp., Pestalotiopsis sp., and Phialophora sp. A representative set of 22 fungal endophyte isolates was selected from the pool of isolates and subjected to large-scale cultivation, followed by extraction of their bioactive metabolites through liquid-submerged fermentation. The resulting crude extracts were evaluated for their inhibitory potential against two Gram-positive bacteria, namely Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA); and two Gram-negative bacteria, namely Escherichia coli and Multi-drug resistant Pseudomonas aeruginosa (MRPA), using the disc diffusion assay. The results indicate that the crude extracts obtained from endophytic fungi colonizing D. philippinensis represent a promising source of bioactive metabolites that exhibit noteworthy inhibitory activity against S. aureus, E. coli, and MRSA, with an effective concentration of 10 mg/mL. This study demonstrates that the fungal endophytes associated with Dillenia philippinensis foliage represent a rich source of bioactive metabolites with significant inhibitory activity against Gram-positive and Gram-negative bacteria. These lead to exploring the potential of these fungal endophytes as a viable source of novel therapeutics.
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... Several antibacterial compounds in the Triterpenoid group also have been reported from other species of the Dillenia genus, such as betulinic acid from the roots of D. suffruticosa [5]. Betulinic acid is also contained in D. phillippinensis and other compounds 3-oxoolean-12-en-30-oic acid, which is also antibacterial active [20]. Nick et al. (1994) [3] reported dillenia A, dillenia acid B, dillenia acid C, dillenia acid D, dillenia acid E, betulinic acid and aldehyde betulin of D. pappuana and showed activity against bacteria against Bacillus subtilis, E. coli and Micrococcus luteus. ...
Antibacterial Compound from n-Hexane Fraction of Dillenia ochreata Leaves
Article
Full-text available
  • Jun 2023
Semprawang (Dillenia ochreata) belongs to the Dilleniaceae family that has been used by the Musi tribe, Banyuasin, South Sumatra, for scurvy medicine. This study aims to isolate secondary metabolites from D. ochreata leaves n-hexane extract and test their antibacterial activity against Escherichia coli and Staphylococcus aureus. The D. ochreata leaves were extracted through the maceration method with n-hexane solvent, and the isolated compounds were purified using column chromatography. The isolated compounds were analyzed using FT-IR, 1H-NMR, and 13C-NMR spectroscopy and compared the spectroscopic data with the literature. The antibacterial activity was determined against the E. coli and S. aureus bacteria with the disc diffusion method and MIC value was determined by the microdilution method. Based on the analysis of the spectroscopic data and compared with literature data, it is suggested that the isolated compounds are 3β-glucopyranosyl-lup-20(29)- en-28-oic, which mixes with aromatic compound. The isolated compounds showed antibacterial activity with a minimum inhibitory concentration (MIC) to E. coli at 120 μg/mL and S. aureus at 60 μg/mL
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Molecular docking and ADMET studies of Dillenia philippinensis Rolfe ( Dilleniaceae ) plant compounds as anti‐inflammatory agents in prostanoid and NF‐kB pathways
Article
  • Dec 2024
The anti‐inflammatory properties of reported compounds from Dillenia philippinensis , also known as katmon, were determined based on their affinity to IRAK‐4, NIK, PLA2, and COX‐2 via molecular docking to determine their capability to block the active site through binding. The binding affinity of the compounds was determined through molecular docking using Autodock Vina. The physiochemical and pharmacokinetic properties were determined using ADMETlab 2.0. Key findings show that tiliroside exhibited the highest binding affinity with COX‐2, PLA2, IRAK‐4, and NIK. Moreover, both tiliroside and the potent inhibitors for each enzyme were found to commonly interact through hydrogen‐bonding and hydrophobic interactions with amino acid residues Val335, Tyr341, Arg499, and Val509 in COX‐2; Ala18, Leu22, His47 in PLA2; Ala211, Val246, Tyr262, Met265, Arg273, and Leu318 in IRAK‐4; Leu406 in NIK. Based on ADMET profiling, they possess moderate to excellent absorption and metabolism and low toxicity despite their low clearance and poor distribution. Betulinic and maslinic acid were found to possess acceptable oral bioavailability according to Lipinski's Rule of Five. Investigation of the binding affinity and druglikeness of the compounds revealed their anti‐inflammatory potential as natural therapeutics. Further experimental studies should be conducted to validate their potency and the initial in silico screening results.
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BIOANALYTICAL METHOD VALIDATION FOR DETERMINATION OF ROSMARINIC ACID IN SIMULATED BIOLOGICAL MEDIA USING HPLC
Article
Full-text available
  • Mar 2021
Objective: This study aims to develop and validate a simple, rapid and accurate HPLC method for the determination of rosmarinic acid (RA), an active marker of Thunbergia laurifolia (TL) tea, in Hank's Balanced Salt Solution (HBSS) using HPLC. Methods: The separation was performed using a Xterra® C18 reversed-phase column (150 3.9 mm, 5 µm). The mobile phase consisted of 0.5% (v/v) glacial acetic acid (A) and acetonitrile (B). The flow rate was 1 ml/min and detection was carried out at 330 nm with UV-visible spectrophotometer. The method was validated according to the US FDA guidance on bioanalytical method validation in 2018. Results: The method was successfully validated in the range of 50-500 ng/ml of RA. Intra-and inter-day precision ranged from 1.6 to 2.1% and 1.4 to 4.5%, respectively. Intra-and inter-day relative errors (% bias) were less than 7.6 and 3.6%, respectively. In addition, it was found that the stability of RA in HBSS could be dependent on the pH and temperature. Conclusion: The developed method met the validation requirements and could be further applied to the permeability study of RA using an in vitro Caco-2 cell monolayer model.
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Betulinic Acid Triggers CD95 (APO-lIFas)- and p53-independent Apoptosis via Activation of Caspases in Neuroectodermal Tumors
Article
Full-text available
  • Dec 1997
Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-XL conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.
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Antibacterial Triterpenoid Acids from Dillenia papuana
Article
  • Oct 1994
Three new oleanene-type triterpenoids, dillenic acids A [1], B [2], and C [3], and two known compounds, 3-oxoolean-1,12-dien-30-oic acid [4] (a new natural product) and the lupene derivative betulinaldehyde, have been isolated from the Papua New Guinean medicinal plant Dillenia papuana. The structures of the new compounds were elucidated as 2 alpha-hydroxy-3-oxoolean-12-en-30-oic acid, 2-oxo-3 beta-hydroxyolean-12-en-30-oic acid and 1 alpha-hydroxy-3-oxoolean-12-en-30-oic acid. The 1H- and 13C-nmr data of all new compounds were assigned unambiguously using a variety of 2D nmr experiments including 1H-1H-COSY, HMQC, HMBC, and NOESY experiments. Of these compounds, 1-4 showed antibacterial activities against Bacillus subtilis, Escherichia coli, and Micrococcus luteus.
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Antibacterial Triterpenoids from Dillenia papuana and Their Structure- Activity Relationships
Article
  • Jan 1996
  • PHYTOCHEMISTRY
Two new oleanene-type triterpenoids, dillenic acids D and E, have been isolated from the leaves and stems of Dillenia papuana together with the new natural product 3-oxoolean-12-en-30-oic acid. Together with these compounds, the known compound, betulinic acid (3 beta-hydroxy-20(29)-lupen-28-oic acid) was isolated as the major component of the fractions studied. Dillenic acids D and E were characterized as 2,3-seco-2-oxoolean-12-en-3-methylester-30-oic acid and 1 alpha,3 beta-dihydroxyolean-12-en-30-oic acid and their nuclear magnetic resonance data were unambiguously assigned using two-dimensional nuclear magnetic resonance techniques. A comparison of antibacterial activities of these compounds with the earlier reported dillenic acids A-C indicated that, aside from a double bond in gamma- or delta-position to a carboxylic group, a ketone function in ring A of an oleanene-skeleton may be required for the observed activity.
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Anti-AIDS agents--XXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives
Article
  • Dec 1997
  • BIOORGAN MED CHEM
Two series of lupane-type triterpenoic acid derivatives were synthesized and evaluated for their inhibitory activity against HIV-1 replication in acutely infected H9 cells, based on the fact that betulinic acid (1) and dihydrobetulinic acid (9) were identified as anti-HIV agents. Among the derivatives, 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (3) and 3-O-(3',3'-dimethylsuccinyl)-dihydrobetulinic acid (11) both demonstrated extremely potent inhibitory activity with EC50 values of < 3.5 x 10(-4) microM, and remarkable in vitro therapeutic index (TI) values of 20,000 and 14,000, respectively. 3-O-(3',3'-dimethylglutaryl)-betulinic acid (4) and-dihydrobetulinic acid (12), 3-O-diglycolyl-betulinic acid (5) and -dihydrobetulinic acid (13) and 3-O-glutaryl-betulinic acid (6) were also potent inhibitors of HIV replication with EC50 values ranging from 0.04 to 2.3 x 10(-3) microM and TI values from 292 to 2344. In addition, compounds 11 and 12 were also active against HIV replication in a monocyte cell line and in peripheral blood mononuclear cells. Our in vitro assay indicated that these compounds are not inhibitors of HIV-1 reverse transcriptase, whereas they inhibited syncytia formation completely in a concentration range of 20-40 micrograms/mL. However, 3-O-(2',2'-dimethylsuccinyl)-betulinic acid (2) was also found to be an inhibitor of HIV-induced membrane fusion with an IC100 value of 20 micrograms/mL, though it displayed significantly lower anti-HIV activity than foregoing compounds with an EC50 value of 2.7 microM and TI of 6.7. Further study is underway to determine the mechanisms of action of these compounds.
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Betulinic acid induces apoptosis in human neuroblastoma cell lines
Article
  • Nov 1997
  • EUR J CANCER
Neuroblastoma has long been recognized to show spontaneous regression during fetal development and in the majority of stage 4s infants < 1 year of age with disseminated disease. Stage 4s disease regresses with no chemotherapy in 50% of the patients. The mechanism by which this occurs is not understood but may be programmed cell death or apoptosis. Betulinic acid (BA) has been reported to induce apoptosis in human melanoma with in vitro and in vivo model systems. Melanoma, like neuroblastoma, is derived from the neural crest cell. We hypothesised that neuroblastoma cells have the machinery for programmed cell death and that apoptosis could be induced by betulinic acid. Nine human neuroblastoma cell lines were treated in vitro with BA at concentrations of 0-20 micrograms/ml for 0-6 days. Profound morphological changes were noted within 3 days. Cells withdrew their axonic-like extensions, became non-adherent and condensed into irregular dense spheroids typical of apoptotic cell death (ED50 = 14-17 micrograms/ml). DNA fragmentation analysis showed ladder formation in the 100-1200 bp region in 3/3 neuroblastoma cell lines treated with BA for 24-72 h. Thus, apparently BA does induce AP in neuroblastoma in vitro. This model will be utilised to investigate the role of apoptosis-related genes in neuroblastoma proliferation and to determine the therapeutic efficacy of BA in neuroblastoma in vivo.
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Betulinic Acid-Induced Apoptosis in Glioma Cells: A Sequential Requirement for New Protein Synthesis, Formation of Reactive Oxygen Species, and Caspase Processing
Article
  • Jul 1999
Betulinic acid (BA), a pentacyclic triterpene, is an experimental cytotoxic agent for malignant melanoma. Here, we show that BA triggers apoptosis in five human glioma cell lines. BA-induced apoptosis requires new protein, but not RNA, synthesis, is independent of p53, and results in p21 protein accumulation in the absence of a cell cycle arrest. BA-induced apoptosis involves the activation of caspases that cleave poly(ADP ribose)polymerase. Interactions of death ligand/receptor pairs of the CD95/CD95 ligand family do not mediate BA-induced caspase activation. BA enhances the levels of BAX and BCL-2 proteins but does not alter the levels of BCL-xS or BCL-xL. Ectopic expression of BCL-2 prevents BA-induced caspase activation, DNA fragmentation, and cell death. Furthermore, BA induces the formation of reactive oxygen species that are essential for BA-triggered cell death. The generation of reactive oxygen species is blocked by BCL-2 and requires new protein synthesis but is unaffected by caspase inhibitors, suggesting that BA toxicity sequentially involves new protein synthesis, formation of reactive oxygen species, and activation of crm-A-insensitive caspases.
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Betulinic acid: A new cytotoxic agent against malignant brain-tumor cells
Article
  • Aug 1999
Malignant brain tumors are the most common solid tumors in children. The overall prognosis for this group of patients is still poor, emphasizing the importance of more effective therapies. Betulinic acid (Bet A) has been described as a novel cytotoxic compound active against melanoma and neuroblastoma cells. Here we report that Bet A was active against medulloblastoma and glioblastoma cell lines. In addition, Bet A exerted cytotoxic activity against primary tumor cells cultured from patients in 4 of 4 medulloblastoma-tumor samples tested and in 20 of 24 glioblastoma-tumor samples. Since a small percentage of primary-glioblastoma-tumor cells (4/24) did not respond to Bet-A treatment, resistance to Bet A might occur. Induction of apoptosis by Bet A involved mitochondrial perturbations, since inhibition of the mitochondrial permeability transition by the mitochondrion-specific inhibitor bongkrekic acid (BA) reduced Bet-A-induced apoptosis. In addition, mitochondria undergoing Bet-A-induced permeability transition triggered DNA fragmentation in isolated nuclei. Cytochrome c was released from mitochondria of Bet-A-treated cells, and might be involved in activation of caspases. Following treatment with Bet A, caspase-8, caspase-3 and PARP were proteolytically processed. Inhibition of caspase cleavage by the broad-range caspase inhibitor zVAD.fmk strongly reduced Bet-A-induced apoptosis, indicating that apoptosis was mediated by activation of caspases. Since Bet A did not exhibit cytotoxicity against murine neuronal cells in vitro, these findings suggest that Bet A may be a promising new agent for the treatment of medulloblastoma and glioblastoma cells that clearly warrants further pre-clinical and clinical evaluation.
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