Temu Kunci

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Procedia Chemistry 13 (2014) 13 37
International Seminar on Natural Product Medicines, ISNPM 2012
Boesenbergia pandurata Roxb., An Indonesian Medicinal Plant:

Phytochemistry, Biological Activity, Plant Biotechnology
Agus Chahyadi a,b, Rika Hartatia, Komar Ruslan Wirasutisnaa, Elfahmia*
a
Pharmaceutical Biology Research Group, School of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10 Bandung, Indonesia, 40132
b
Pharmacy Department, Faculty of Pharmacy, Universitas Haluoleo, Kendari, 93231, Indonesia
Abstract
Boesenbergia pandurata Roxb. (Zingiberaceae), known as temu kunci, is one of the Indonesian medicinal plants. Its rhizome
has been traditionally used in folk medicine for treatment of several diseases. Rhizome of B. pandurata contains essential oils
and many flavonoid compounds that showed many interesting pharmacological activities, such as antifungal, antibacterial,
antioxidant, etc. Interestingly, this plant has several prenylated flavonoid compounds, panduratins, that showed very promising of
biological activities, especially as strong antifungal and antibacterial, anti-inflammatory, and anti-cancer. This paper aims to
review chemical constituents of this plant and their pharmacological activities and also to give a brief view through
biotechnological perspective concerning the several possibilities to produce several valuable prenylated flavonoids from this
plant.
2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
2014 The Authors. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Peer-review under responsibility of the School of Pharmacy, Bandung Institute of Technology.
Peer-review under responsibility of the School of Pharmacy, Bandung Institute of Technology
Keywords: Boesenbergia pandurata, Essential oil, Prenylated flavonoid, Panduratin, Antibacterial
* Corresponding author. Tel.: +62-22-2504852 fax: +62-22-2504852

E-mail address:elfahmi@fa.itb.ac.id
1. Introduction
Boesenbergia pandurata Roxb. Schlecht. (Zingiberaceae) is one of the ginger plants that is found in South East
Asia. In Indonesia, this geophytic plant is known as temu kunci, grows wildly in teak forests, and is cultivated
everywhere. This plant has many synonymously botanical names, such as Gastrochilus panduratum RIDL.,
Kaempferia pandurata Roxb., Curcuma rotunda L., and Boesenbergia rotunda Linn. Mansft 1-3. According to the
Indonesian medicinal plants literatures, fresh rhizome of B. pandurata has been long time utilized as spice,
particularly, the young rhizome was very popular for seasoning vegetables. It was strongly believed that its efficacy
could strengthen the stomach. As a traditional medicine, the sliced rhizomes which are chewed together with areca
1876-6196 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Peer-review under responsibility of the School of Pharmacy, Bandung Institute of Technology
doi:10.1016/j.proche.2014.12.003
14 Agus Chahyadi et al. / Procedia Chemistry 13 (2014) 13 37
nut (Areca catechu) could treat dry cough and aphtha1,2. As food, in the form of porridge, combination between
rhizome and Pimpinella anisum was used to treat stomach distended and as a diuretic for children, while
combination with coconut milk was used as anti-anthelmintic. Rhizome of B. pandurata was also used as a
traditional medicine for treatment inflammation in women uterus and in combination with other spices, for treatment
vaginal infection 2. The rhizome of this plant is a one of the components of herbal medicine jamu in Indonesia 4. In
the references of herbal preparations published by The National Agency of Drug and Food Control (NA-DFC or
BPOM Republik Indonesia), herbal preparation from rhizome of B. pandurata is utilized as an anti-inflammatory 5
and an anti-cancer 6.
This plant has been identified to contain various essential oils (EOs) and also several flavonoid compounds that
have demonstrated many biological activities. Most of its flavonoids have unique features with some prenyl
substituents integrated in their main structures 7,8. A panduratin derivative are prenylated flavonoids from B.
pandurata that showed broad range of biological activities, such as strong antibacterial acitivity9-11, anti-
inflammatory 12, and anti-cancer 13.
The aim of this paper is to present information of the chemical constituents and also to compile various
pharmacological studies that is well established from this plant. A second aim is to give a brief view concerning the
possibility to produce biological active compounds from B. pandurata through biotechnological approach. All of
literatures have been cited from both offline and online media. Offline literatures were based on Handbooks of
Indonesian Medicinal Plants, Indonesian Herbal Pharmacopeia, and Herbal Preparations of NA-DFC. Whereas ISI
Web, Scopus, Pubmed, and other online media were used to regain any online publications such as scientific
journals, patents, etc. All of them were used to compile this paper.
2. Phytochemistry
2.1. Flavonoid
Flavonoids are large secondary metabolites found in rhizome of B. pandurata. More than 51 flavonoid
compounds from B. pandurata have been isolated and their structure was confirmed. However, only three classes of
flavonoid have been reported exist in B. pandurata rhizome. The main flavonoids are chalcones, flavanones, and
flavones, classified according to their skeletons (Fig. 1). However some of these flavonoids also exist in other plants.
Most of them showed a unique structure with some prenyl substituents integrated in their main skeleton.
Interestingly, more than half of total flavonoids isolated from B. pandurata are prenylated flavonoids. But only two
classes of flavonoids have prenylated derivative, namely prenylated chalcones and prenylated flavanones. Flavones,
however, there is no report that showed the existence of their prenylated derivative in B. pandurata.
Fig. 1 Typical class of regular flavonoids in B. pandurata
2.1.1. Unprenylated flavonoids
Several known flavonoid chalcones, flavanones, and flavones have been isolated and identified from rhizome
extract of B. pandurata. In our review, there are five chalcones and two hydrochalcones, due to the difference of
their oxygenation pattern, have been reported (Fig. 2). They are cardamonin (1), a known 2,4-dihydroxy-6-
Agus Chahyadi et al. / Procedia Chemistry 13 (2014) 13 37 15
methoxy chalcone 14 and a major chalcone in B. pandurata rhizome 15; pinocembrin chalcone (2), a known 2,4,6-
trihydroxychalcone 15; helichrysetin (3), a known 2,4,4-trihydroxy-6-methoxychalcone 16; 2,6-dihydroxy-4-
methoxychalcone (4) 14; flavokawain C (5), a known chalcone possessing a 4-oxygenated pattern of A-ring namely
2,4-hydroxy-4,6-dimethoxychalcone 17; and also two known hydrochalcones, namely 2,4,6-trihydroxychalcone
(6) and uvangoletin (7) 12. Structural analysis showed that oxygenation pattern in most of these chalcones, except
compound 3 and 5, only occurred at B-ring.
Fig. 2 Regular chalcones in B. pandurata
Flavanones are isomerized products from chalcones. The isomerise of chalcones to form flavanones are naturally
due to the presence of chalcone isomerase and also this process still occurs spontaneously even in the absence of this
enzyme. Six known flavanones have been isolated from B. pandurata, namely pinostrobin (8), the major flavanone
in B. pandurata; pinocembrin (9) 14; 5,7-dimethoxyflavanone (10) 18; alpinetin (11) 19; sakuranetin (12) 7; and 7,4-
dihydroxy-5-methoxyflavanone (13) 17 (Fig. 3). In the Indonesian Herbal Pharmacopeia, compound 8 is used as a
marker compound in examination of metabolite profile from botany, extract and herbal preparations of B. pandurata
rhizomes 20.
Fig. 3 Regular flavanones in B. pandurata
Some flavones are also found in black rhizome, another variety of B. pandurata with yellow rhizomes. They are
formed through the dehydrogenation and followed by the formation of double bound in C2-C3 of flavanones. There
are eight known flavones contained in black rhizome of B. pandurata 18. Tectochrysin (14), 5,7-dimethoxyflavone
(15), and 5-hydroxy-3,7-dimethoxyflavone (16) are flavones with no oxygenated pattern in A-ring, while the others,
namely 5,7,4-trimethoxyflavone (17), 5-hydroxy-7,4-dimethoxyflavone (18), 5,7,3,4-tetramethoxyflavone (19),

5-hydroxy-3,7,4-trimethoxyflavone (20), and 5-hydroxy-3,7,3,4-tetramethoxyflavone (21), have oxygenated
pattern in their A-ring. However, only compounds 16, 20, and 21 have methoxy constituents in C3 position (Fig. 4).
According to this report, the chemical constituents in black rhizome differ substantially to B. pandurata with
yellow/red rhizomes, the main plant that is reported in this review. So far, only compound 14 that was successfully
isolated again in yellow rhizomes 17, the rest, there is no more report that showed these flavones were isolated again.
Fig. 4 Regular flavones in B. pandurata
2.1.2. Prenylated flavonoids
Unlike the other Zingiberaceae plants, most of the flavonoids from B. pandurata are very distinctive due to the
prenyl substituents in their main skeleton. More than 31 prenylated flavonoids have been isolated from rhizome of B.
pandurata (Fig. 5). Boesenbergin A (22) is a first prenylated flavonoid isolated from this plant by
Tuntiwachwuttikuls group 14. This flavonoid with chalcone skeleton has three oxygenated aromatic carbon pattern
at B-ring, also, a geranyl moiety at C5 aromatic then interacted with oxygen at C6 aromatic to form a pyran ring. In
the same pattern, prenylation also occurred at C3 aromatic then finally generated a compound boesenbergin B (23),
first isolated by Mahidol et al. 21. Also, both of these compounds, through acid-catalyzed cyclization, have been
successfully chemically synthesized 14,21. Still in the same group, two prenylated flavonoids, panduratin A (24) and
rubranine (25), were also isolated by Tuntiwachwuttikul et al. 22. Compound 25 is a known prenylated chalcone
previously reported from Brazilian Tree, Aniba rosaeodora (Lauracea). Whereas compound 24 is a new prenylated
flavonoid built from chalcone skeleton with 3 oxygenated patterns only at B ring and a geranyl substituent at C2-C3
position through Diels-Alder cycloaddition. This compound is a major prenylated chalcone in B. pandurata 15. Two
compounds, that their structures are closely related with compound 24, were also reported by Pandji et al. 19 and re-
confirmed by Win et al. 17 as (-)-isopanduratin A2 (26) and ()-isopanduratin A1 (27). These two compounds have a
methoxy substituent at C2 aromatic, while in compound 24 at C4 aromatic. Also, the difference between compound
26 and 27 is only in their chirality.
A new prenylated chalcone with a cyclohexene moiety analogous to compound (24) was successfully isolated
from methanol extract of fresh rhizome B. pandurata. This compound is 4-hydroxypanduratin A (28), where the
substituent at C4 aromatic is hydroxyl moiety 15. Another new cyclohexenyl chalcone analogous to compound 24
and 28 named panduratin C (29), a prenylated chalcone with methoxyl moiety at C6 (B ring) and 3 hydroxyl
moieties at C2, C4 (B ring) and C4 (A ring), has been isolated from this plant 16. Two new prenylated chalcones
from B. pandurata were also reported, namely 2,4-dihydroxy-3-(1-geranyl)-6-methoxychalcone (30) and
(1R,2S,6S)-2-hydroxyisopanduratin A (31), and several known prenylated chalcones, namely ()-6-
methoxypanduratin A (32), and (-)-nicolaioidesin B (33) 17.
Fig. 5 Prenylated chalcones in B. pandurata

Application of chiral columns were able to separate similar compounds with the difference of optical active
rotation from B. pandurata. Morikawa et al. 23, using a Ceramospher chiral column, was able to separate some of
compounds (+)-panduratin A (24) and (-)-panduratin A (34), (+)-4-hydroxypanduratin A (27) and (-)-4-
hydroxypanduratin A (35), and (+)-isopanduratin A (36) and (-)-isopanduratin A (37). Several new prenylated
chalcones were also successfully isolated using this column, namely (+)-krachaizin A (38), (-)-krachaizin A (39),
(+)-krachaizin B (40) and (-)-krachaizin B (41). The pattern of prenylation and oxygenation which occurred to these
compounds is similar to the compound 24. The difference is only in the rearrangement of the cyclohexene (geranyl)
moiety.
Some prenylated flavanones, although not as many as prenylated chalcones, were also reported exist in the B.
pandurata rhizome. There are 10 compounds that have been confirmed by several authors (Fig. 6). Win et al. 17
successfully isolated a new compound, (2R)-8-geranylpinostrobin (48), together with three known compounds, i.e.
(2S)-6-geranylpinostrobin (49), (2S)-7,8-dihydro-5-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-8-phenyl-2H,6H-
benzo[1,2-b:5,4-b]dipyran-6-one (50), and (-)-6-geranylpinocembrin (51). Four new compounds were also reported
by Morikawa et al. 23, two diastereomer rotundaflavones Ia (52) and Ib (53), and IIa (54) and IIb (55), together with
2 known compounds, namely, 5,7-dihydroxy-8-geranylflavanone (56) and 7-methoxy-5-hydroxy-8-geranylflavanone
(57). The prenylation patterns in these flavanones occurred only in aromatic B-ring at C6 and/or C8 position. In
addition, there are no Diels-Alder products as in most of prenylated chalcones.
Fig. 6 Prenylated flavanones in B. pandurata
2.2. Essential oils (EOs)
As found many ginger plants, B. pandurata also contains many essential oils that have been reported their
structure and biological activities. The EOs of B. pandurata consist of largely amount of oxygenated and non
oxygenated monoterpenes. The major compounds of EOs that were isolated by various methods and solvents were -
terpinene (58), geraniol (59), camphor (60), -ocimene (61), 1,8-cineole (62), myrcene (63), borneol (64), camphene
(65), methyl cinnamate (66), terpineol (67), geranial (68) and neral (69) (Fig. 7) 19,25-27. Some EOs were also
presented but in very small amounts, such as nerolidol, citral, limonene, and 11-dodecen-1-ol 26.
Fig. 7 The major compounds of EOs in B. pandurata
2.3. Miscellaneous compounds
Several non-flavonoid compounds were also found in B. pandurata rhizome. These compounds are derived from
the class of dehydrokawain, phenylbenzoic acid, and prenypropanoid (Fig. 8). They are dihydro-5,6-dehydrokawain
(70) 7 and 5,6-dehydrokawain (71), a new prenylated phenylbenzoic acid, geranyl-2,4-dihydroxy-6-
phenethylbenzoate (72) 17, and a known compound, 2,4-dihydroxy-6-phenethylbenzoic acid methyl ester (73) 23.
Two new compounds from prenylated prenylpropanoids, panduratin H (74) and I (75), were also found in rhizome of
B. pandurata 8.
Fig. 8 Other compounds in B. pandurata

3. Biological Activity
Zingiberaceae family has been widely known for their medicinal and economic potency. Many species are used
as sources of indigenous medicines, vegetables, food flavors, spices, dyes, condiments as well as ornamentals 28.
Like other gingers, B. pandurata has been used for long time ago as traditional medicine to treat stomach discomfort,
diarrhoea, colic disorder, dry cough, mouth iritation, dental carries, leukorrhea, rheumatism, fungal infection, and
has also been used as anti-septic for wound, anti-insecticidal, anti-mutagenic, anti-tumour, and anti-inflammatory
1,2,7,13,14,16,19,29
.
According to its usages as traditional medicine, many researchers have investigated its biological activities. Both
its extract and its compounds showed many interesting activities, particularly panduratin derivatives that showed
strong antibacterial activity.
3.1. Antioxidant activity
Compound 24 (panduratin A) has significant protective ability against oxidative damage caused by reactive
intermediates. Sohn et al. 30 examined the ability of 24 to protect hepatocytic injury in a human hepatoma (HepG2)
cell induced by tert-Butylhydroperoxide (t-BHP) and characterized the mechanism involved. They found that 24
reduced the cell growth suppression caused by t-BHP and pretreatment with 10-15 M of 24 significantly increased
the cell viability. As comparison, at the same concentration (15 M), 24 restored cell viability a little bit higher than
sylibin, a natural remedy for liver diseases from Silybum marianum that has been used for centuries. In the cellular
mechanism, 24 could restore the formation of MDA (malondialdehyde) caused by lipid peroxides activity and the
depletion of intracellular GSH (glutathione) in t-BHP-treated HepG2 Cells. The overproduction of reactive oxygen
species (ROS) caused by t-BHP was also reduced by incubation with 24 in a dose-dependent manner and this
reductive effect was also enhanced with time. These results indicate that 24 is probably able to scavenge radical
species in t-BHP-treated cells.
Still in the related mechanism, several flavonoids of B. pandurata, due to their antioxidant property, also showed
inhibitory activities against the lipid peroxidation and neuroprotective effects against L-Glutamate toxicity in rat
brain N18-RE-105 Cells 31. Compounds 1, 4, 8, 9, 24, and 28 were evaluated for their inhibitory activities against
lipid peroxidation and they found that compounds 24 (IC50 = 15 M) and 28 (4-hydroxypanduratin A, IC50 = 4.5
M) showed stronger inhibitory activities even than (+)-catechin (IC50 = 17 M). Analysis of structure-activity
relationships (SAR) showed that: (i) hydroxyl moiety at C4 aromatic plays an important role for the activity
compared with methoxyl moiety as observed in 9 (IC50 = 210 M) toward 8 (IC50 = 230 M); (ii) the chalcone
skeleton with a bouble bond strongly affects for a higher activity than flavanone as shown in 4 (IC50 = 70 M)
toward 8 (IC50 = 230 M); (iii) and the most significant is geranyl substituent that generated much higher activity in
28 (IC50 = 4.5 M) than 1 (IC50 = 38 M) as a non-prenylated chalcone. In addition, compounds 8, 9, 24, and 28 also
exhibited more significant neuroprotective effect in L-Glutamate-treated N18-RE-105 Cells with EC50 values 37, 48,
13, and 14 M, respectively, than (+)-catechin (EC50 = 160 M). Interestingly, there were no compounds that
showed cytotoxicity against the cells even at 50 M.
Compound 22 also displayed potent antioxidant activity against oxygen radical. Using oxygen radical absorbance
capacity (ORAC) assay, 22 was found to possess antioxidant capacity at 20 g/mL which was equivalent to a 11.91
M Trolox, a vitamin E analogue 32. It seems that the prenyl substituent in 22, 24 and 28 might highly contribute for
higher antioxidant activity and also assist these compounds to enhance their transmembrane transport into the cells
and their interaction with the target proteins (Botta et al., 2005; Shen et al., 2012).
3.2. Anti-inflammatory
Eight flavonoid compounds isolated from rhizome of B. pandurata were assayed for their inhibitory activity
against inflammation 7,12,32,33. Compounds 24 and 28 were evaluated for topical anti-inflammatory activity using ear
edema in rats as the experiment model. 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 4 g/ear) was used as an
inflammogen to provoke edematous response. The results showed, pretreatment of the rat ear by topical application
of compound 24 or 28 (20-2000 g/ear) significantly inhibited TPA-induced ear edema formation in a dose-
dependent manner and the ID50 values were 84 and 12 g/ear, respectively 7.
Compounds 1, 6, 7, 24, 28, and 29 were also evaluated for their inhibitory activities against excessive
production of the inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2) and tumour necrosis
factor- (TNF-), that are commonly involved in various pathophysiological processes including inflammation and
carcinogenesis 12. Using an inflammogen lipopolysaccharide (LPS), they found that, the production of NO in LPS-
treated RAW264.7 cells was strongly inhibited by 24 (IC50 = 5.3 M), 28 (IC50 = 13.3 M), and 1 (IC50 = 24.7 M).
The others, however, showed only moderate or mild inhibitory activity. Compared with L-Nitroarginine (L-NA, IC50
= 61.8 M) and caffeic acid phenethylester (CAPE, IC50 = 5.6 M), it seems that 24 will be a new promising agent
as NO inhibitor. Not only strongly inhibited NO production, compounds 24 and 28 also demonstrated strong
inhibition against PGE2 production (IC50 = 10.5 and 12.3 M) and moderate activity on TNF- production (IC50 =
60.3 and 57.3 M), respectively. Previously, the potent inhibition of 24 on NO and PGE2 production in LPS-treated
RAW264.7 cells was actually reported by Yun et al. 33. They strongly proposed that the inhibitory effect of 24 is due
to its activity to suppress the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)
enzyme and activation of NF-KB.
Recently, another prenylated chalcone, 22, has been also reported could inhibit the NO production in IFN-
/LPS -treated RAW264.7 cells. Compound 22 significantly decreased the NO level in a dose dependent manner. At
50 g/mL, 22 was able to decrease the NO level from 36.68 to 25.69 M without any toxicity effect on the
RAW264.7 cells 32.
Apparently, unlike in their antioxidant properties, the methoxyl moiety at C4 aromatic of 24 is very essential for
higher anti-inflammatory activity compared with the hydroxyl moiety, as observed in 28. The rest, such chalcone
skeleton, ,-double bond and prenyl substituent, are still essential for activity 12.
The potent anti-inflammatory and hepatoprotective agents were exhibited by some compounds. Compounds 1, 4,
11, 28, 35, 36, 37, 40 and 41 showed potent inhibitory activity in TNF--induced cell death in L292 cells. All of
them had IC50 values of 7 to 30 M 23.
3.3. Antifungal activity
The chloroform extract of B. pandurata rhizome was tested for its antifungal activity against clinical isolates of
Candida albicans, Cryptococcus neoformans and Microsporum gypseum. According to the minimum inhibitory
concentration (MIC) values, the chloroform extract exhibited strong activity against both C. neoformans and M.
gypseum (MIC = 64 g/mL), but weak against C. albicans (MIC > 512 g/mL). These results confirm the usage of
B. pandurata as a promising candidate for the development of a remedy for opportunistic fungal infections in AIDS
patients, since all of the fungal above commonly cause infection in HIV patients 34.
Due to its antifungal activity, the extract of B. pandurata was also investigated for their potential use as
fungicides in the agriculture. The ethyl acetate extract could inhibit mycelia growth of Phytopthora capsici, a
soilborne pathogen causing epidemic disease in many crops. Nine fractions of ethyl acetate extract revealed anti P.
capsici about 300 fold more efficient than captan (100,000 ppm) as an antifungal control. The concentrations
required to inhibited the mycelia growth of P. capsici by 90% (ED90) were about 300 ppm for each fraction 35.
Traditionally, B. pandurata was utilized to treat some of diseases related to the mouth and dental problem, such
as irritation and dental carries. Therefore, the ethanol extract of B. pandurata was investigated for its effect on
adhesion of Candida albicans to denture acrylic surface, since this fungus is found in the oral cavity, usually
associated with denture stomatitis, adheres to oral surface and finally determines in the development of mouth/teeth
infection. They found that the ethanol extract was able to reduce the adhesion of both C. albicans 13803 and clinical
isolate strain in a dose dependent manner. Pretreatment with 100 mg/mL extract siginificantly reduced the number of
adherent yeast by approximately 75%. As a comparison, 0.2% of chlorhexidine gluconate, a positive control,
exhibited more than 90% inhibitory effect 36. Hence, the utilization of B. pandurata may be useful to treat candida-
associated denture stomatitis and help to prevent recurrence of the infection.
3.4. Antibacterial activity
There are many reports concerning antibacterial properties of B. pandurata. The crude extracts, essential oils
(EOs), and also the purified compounds of B. pandurata have been intensively investigated for their antibacterial
activity. Interestingly, all of them showed strong activity against various strains of bacteria even in low
concentration. All of the assayed bacteria covered foodborne pathogenic bacteria, cariogenic bacteria, acne-causing
bacteria, both clinical Staphylococcus and Enterococci isolates.
The ethanol extract and EOs of B. pandurata were reported to possess antibacterial activity against foodborne
pathogenic bacteria. At the concentration about 5 to 10% (v/v), the extract inhibited all of Listeria monocytogenes (5
strains) for 24 h, while the EOs inhibited all strains of L. monocytogenes and 4 strains of Salmonella at 0.4%.
Addition of 0.2% EOs to apple juice reduced 4 log CFU/mL L. monocytogenes Scott A and 2 strains of Salmonella
to an undetectable level within 1 to 2 days 37. The EOs of B. pandurata were also tested to the other foodborne
pathogens, i.e. Eschericia coli, Staphylococcus aureus, Bacillus cereus, and as well as L. monocytogenes. The
hydrodistillated EOs had the highest efficiency against three Gram-positive strains of bacteria (S. aureus, B. cereus,
and L. monocytogenes) with minimum inhibition concentration (MIC) values of 12.5, 12.5, and 6.25 mg/mL,
respectively, while the MIC value for E. coli, a Gram-negative, was 50 mg/mL 26. The mechanism of action EOs of
B. pandurata on bacterial cell was strongly hypothesized due to its ability to change the permeability and to alter salt
tolerance of the cell. In the EOs-treated E. colli K1.1 cells, after exposing 0.22% of EOs, the cell became empty
because the leakage of inorganic compounds (potassium and calcium ion) and organic compounds (nucleic acid and
protein) from bacterial cell 27. These results showed that both extract and EOs of B. pandurata are potent as
pathogen control and natural preservative in food systems.
The methanol extract of B. pandurata exhibited potent antibacterial activity against cariogenic bacterium
Streptococcus mutans. The extract conferred fast killing bactericidal effect against S. mutans in 2 min at 50 g/mL
of extract concentration 38. Compound 36 confirmed the antibacterial activity of the extract in which 36 had MIC of
4 g/mL to inhibit the growth of S. mutans. The anticariogenic activity of 36 was much stronger than other natural
anticariogenic agents such as sanguinarine (12 g/mL) and green tea extract, carvacrol,thymol, isoeugenol and
eucalyptol (125 to 500 g/mL). The MBC of 36 was 8 g/mL and at concentration 20 g/mL, 36 showed fast-killing
in one minute. 36 was also highly active to inhibit S. salivarus, S. sanguis, and S. sobrinus with similar MIC value (4
g/mL) 39. S. mutans, S. sanguis, and Actinomycetes viscous are multi-species oral biofilms causing oral disease
(dental plaque) through the formation of dental biofilm. Compound 24 was found to be able to kill these multi-
species strains and also able to prevent and reduce the formation of biofilms in type III mucin-coated wells. 24
showed a MIC of 1 g/mL and bactericidal activity against a mixture of these multi-species strains at 2 x MIC (4
g/mL) for 8 h. The prevention of biofilm formation was inhibited by > 50% at 8 x MIC (8 g/mL) of 24 and the
reduction effect on 24 h multi-species biofilms was achieved by > 50% after exposure at 10 g/mL of 24 for 15 min.
All of the MIC value, preventive and reducing effect of 24 showed a trend similar both concentration and time
exposure to that of chlorhexidine gluconate 11. Another previous report also showed that 24 had antibacterial activity
against some of the periodontitis-causing bacteria, i.e., Porphyromonas gingivalis, Prevotella intermedia, and P.
loescheii, with MIC values of 4, 2 and 4 g/mL, respectively 40. These results indicate that B. pandurata can be used
for treatment of dental carries and compounds 24 and 36 are very likely responsible for the activity.
Compounds 24 and 36 demonstrated high activity against bacterial causing acne and skin diseases. Both MIC and
minimum bactericidal concentration (MBC) values of 24 and 36 for Propionibacterium acnes, a predominant
bacterium in the skin and plays an important role in the pathogenesis of inflamed lesions, were 2 and 4 g/mL (for
MIC) and 4 and 8 g/mL (for MBC), respectively. At MBC concentration for 48h, the time-dependent killing effect
of 24 and 36 showed maximum inhibition to the growth of P. acnes 41. The powerful antibacterial effect of 24 and 26
against skin microbes might be useful for developing natural skin care products for acne suppression.
Interestingly, compound 24 is highly active against both clinical Staphylococci and Enterococci isolates. The
antibacterial activity of 24 was tested against 108 Staphylococcus strains consisting of methicillin-resistant
Staphylococcus aureus (MRSA, 27 isolates), methicillin-susceptible S. aureus (MSSA, 27 isolates), methicillin-
resistant coagulase-negative staphylococci (MRCNS, 28 isolates), and methicillin-susceptible coagulase-negative
staphylococci (MSCNS, 26 isolates). Although resistant to ampicillin and several commonly used antimicrobials, all
of isolates were significantly inhibited by 2 g/mL of 24. The 24 had a MIC at which 90% of bacteria were
inhibited of 1 g/mL for clinical staphylococcal isolates. The in vitro MBCs of 24 after 24 h incubation
demonstrated that 24 was able to kill staphylococcus strains with MBCs of 8 g/ml for MRSA, MSSA, and MRCNS,
and 4 g/mL for MSCNS 9. Compound 24 was also assayed for its antibacterial properties against 23 isolates of
clinical Enterococcus strains consisting of 10 isolates of Enterococcus faecalis and 13 isolates of E. faecium. These
enterococci are the most common Gram-positive cocci in the intestinal tract and have been documented to cause in
fection of the urinary tract and other sites of humans and have become resistant to ampicillin and erythromycin.
However, all clinical enterococci isolates were found to be susceptible to 24, with MICs of 2 g/mL and MIC90 of
1 g/mL. They were killed by 24 after 24 h incubation with in vitro MBCs of 8 g/mL. In the time-kill assay, the
bactericidal endpoint for clinical enterococci was reached after 30 min of incubation at a concentration 4 x MIC of
24. Compound 24 was also evaluated for its antibiofilm activity to inhibit and to eradicate the growth of biofilm-
producing enterococcal strains. The results demonstrated that the growth of biofilm-producing enterococcal strains
can be inhibited and eradicated by 24 at concentrations of 4 g/mL and 16 g/mL, respectively, and 24 was more
active than daptomycin linezolid 10. This finding showed that the activity of 24 against both clinical Staphylococci
and Enterococci isolates was generally stronger than available antimicrobial agents, such as ampicillin, daptomycin,
erythromycin, gentamicin, levofloxacin, linezolid, oxacillin, tetracycline, thymol, and vancomycin 9,10. Therefore, it
might be very useful to use 24 as a new natural antibacterial agent for treatment several multidrug-resistant strains-
caused diseases.
3.5. Anti-periodontal activity
Periodontitis is an inflammatory disease that affects the supporting tissues of teeth, leading to progressive
destruction of connective tissue attachment and alveolar bone. Periodontitis is initiated by accumulation of microbial
plaque and activation of gingival inflammation through overexpression of matrix metalloproteinases (MMPs),
leading to tissue destruction. Porphyromonas gingivalis, a Gram-negative bacterium, is the one of the oral pathogens
that can trigger periodontal host cells to release inflammatory mediator including cytokines, tumor necrosis factor-
(TNF-), prostaglandin, and MMPs 42,43. Since compound 24 has shown anti-inflammatory activity and antibacterial
activity against periodontitis-causing bacterium P. gingivalis, some of the subsequent studies have been done in
order to analyze the cellular mechanism of action both B. pandurata extract and 24 in P. gingivalis supernatant-
induced human oral epidermoid KB cells and human gingival fibroblast-1 (HGF-1) cells.
The MMP-9 protein expression elevated up to 2.4 fold after exposed with 10% P. gingivalis supernatant in KB
cells, but its overexpression was inhibited up to 43% by 24 at 25 M. At this concentration, 24 was able to decrease
the expression level of MMP-9 mRNA up to 56% and also reduced urokinase plasminogen activator (uPA) mRNA
expression up to 37%. uPA, in the plasmin-dependent pathway, catalyzes the cleavage of plasminogen into the active
plasmin that capable of activating MMP-9, leading to tissue destruction during periodontal inflammation. 24 was
found did not interfere in regulation of the endogenous tissue inhibitor of metallloproteinases (TIMPs) in P.
gingivalis supernatant-induced KB cells 42.
The expression of MMPs was also promoted via activation of mitogen-activated protein kinase (MAPK)
signaling pathways. Several kinases such as extracellular signal-related kinase (ERK1/2), p38 kinase and c-Jun N-
terminal kinase (JNK) were found to be activated by P. gingivalis. Like 24, the ethanol extract of B. pandurata
showed similar patterns in inhibiting the expression of MMP-9 protein and MMP-9 mRNA (percentages of
inhibition at 10 g/mL of extract were 52 and 45%, respectively) and also had no effect on either TIMP-1 or TIMP-2
mRNA expression. The extract of B. pandurata interfered MMP-9 expression in P.gingivalis supernatant-induced
KB cells by down-regulating MAPK phosphorylation. At 10 g/mL, the extract strongly blocked both
phosphorylation of ERK1/2 (80%/72%) and JNK (55%), whereas on p38 phosphorylation was only inhibited up to
18%. The overexpression of Elk1, c-Jun and c-Fos, the transcription factor components for phorphorylation of
ERK1/2, p38 and JNK, were also inhibited by 10 g/mL of B. pandurata extract. The supernatant of P. gingivalis
also stimulated the expression of MMP-9 gene-regulating promoters, i.e activator protein (AP-1) and nuclear factor-
kappa B (NF-B) in KB cells, but interestingly, their expression could be inhibited up to 42 and 38% by extract of B.
pandurata at 10 g/mL, respectively 29. Compound 24 clearly confirmed the mechanism inhibitory activity of the B.
pandurata extract in down-regulating the MAPK phosphorylation (ERK1/2, p38 and JNK), the transcription factors
(Elk1, c-Jun and c-Fos) and activators of MMP-9 gene-regulating promoters (AP-1 and NF-B). Further study
showed that JNK signaling is primarily involved in regulation of MMP-9 gene expression in P. gingivalis
supernatant-stimulated KB cells through up-regulating the AP-1 element. It was proved by using a specific JNK
inhibitor (SP600125, 20 M), the MMP-9 secretion and protein were completely blocked and the activity of AP-1
was also strongly abolished (>65%). Interestingly, 24 was found to completely abolish the expression of ERK1/2
and JNK phosphorylation and to strongly block AP-1 activity(> 80%) in P.gingivalis supernatant-induced KB cells
at 25 M 44. These findings suggest that the mechanism inhibitory effect of 24 in down-regulating MMP-9
expression is through the inhibition of MAPK signaling.
In another P. gingivalis-treated cell, a human gingival fibroblast-1 (HGF-1), another MMP family (MMP-2) was
significantly overexpressed. Similar with MMP-9, MMP-2 is also involved in inflammatory progression, leading to
tissue destruction and bone loss. The ethanol extract of B. pandurata (10 g/mL) significantly suppressed the MMP-
2 in vitro activity, secretion and protein expression. Furthermore, B. pandurata had an inhibitory effect on
transcriptional level by decreasing MMP-2 mRNA expression in P. gingivalis supernatant-treated HGF-1. The
mechanisms of B. pandurata in inhibiting MMP-2 expression was apparently similar to MMP-9 by down-regulating
the expression of all MAPKs phosphorylation (ERK1/2, p38 and JNK). The overexpressed of CREB
phosphorylation, one of the regulatory elements in the promoter regions of the MMP-2 gene, was decreased by B.
pandurata extract in dose dependent manner in HGF-1 cells treated with P. gingivalis supernatant. These results
strongly suggest that B. pandurata extract reduced MMP-2 gene expression via inhibition of the CREB and JNK
signaling pathway 43.
3.6. Antiviral activity
Seven compounds isolated from B. pandurata, 1, 3, 6, 7, 24, 28, and 29, have been tested for their anti-HIV-1
protease activity. Among the isolates tested, 28 exhibited the most potent HIV-1 PR inhibitory with an IC50 value of
5.6 M, followed by 24 (IC50 = 18.7 M), whereas other compounds possessed weak activity (IC50 > 100 M). At
100 M, 1, 29, 3, 6, and 7 had percentage of inhibition of 47.6, 43.1, 14.1, 7.5, and 2.7%, respectively. Compared
with a positive control, acetyl pepstatin (IC50 = 3.4 M), 28 seemly has potency as a new promising for anti-HIV.
According to the SAR of these compounds on anti-HIV-1 PR activity, (i) hydroxyl moiety at position 4 conferred
higher activity as shown in 28 compared with 24, (ii) prenylation of dihydrochalcone was essential for activity as
shown in 28 compared with 6, (iii) hydroxylation at position 4(A ring) reduced activity as shown in 3 compared
with 1, and (iv) introduction of double bond at C1 and C6 of chalcone gave higher activity as shown in 1 compared
with 7 16.
Compounds 24 and 28 were highly active in inhibiting dengue-2 virus NS3 protease. They inhibited activity of
DEN-2 NS2B/3 virus protease more than 65% at 80 ppm. Interestingly, 28, even in lower concentration (40 ppm),
showed inhibition up to 50% and was better than 24 (27%). Other compounds i.e., 1, 8, 9, and 11, however, only
showed low inhibitory activities although at high concentration (>120 ppm). In different condition, combination of 1
and 9 gave inhibition more than 50% at 120 ppm and was higher than in individual inhibitory activity. The
mechanism of inhibitory activities of these compounds on DEN-2 NS2B/3 virus protease revealed that compounds 1
and 8 were non-competitive inhibitor, whereas compounds 24 and 28 were competitive inhibitor, with Ki values of
345, 377, 25, and 21 M, respectively. The small Ki values of the competitive inhibitors, especially 28, show the
potential of the prenylated flavonoids as in vitro inhibitors for the DEN-2 NS2B/NS3 protease 45.
Compound 8, though at 120 ppm only inhibiting more than 50% activity of DEN-2 NS2B/3 virus protease and
therefore thought to have weak activity 45, exhibited interesting antiviral activity toward Herpes Simplex Virus-1
(HSV-1). 8 inhibited HSV-1 replication with 50% effective concentration (EC50) of 22.71 g/mL and had no
cytotoxicity on host cell (HSV-1-infected Vero E6 cell) at 95.37 g/mL. Some interesting findings revealing the
mode of antiviral activity of 8 showed that free virus was much more sensitive to 8 than acyclovir (ACV, a
nucleoside analogue for treatment of HSV-1 infections). Before and during the adsorption of virus into host cells, 8
reduced the HSV-1 virus numbers much more significant than ACV which only showed antiviral activity during the
replication period. By using atomic force microscopy (AFM), the mechanism of viral inactivation by 8 was clearly
seen. In time and dose dependent manner, 8 caused the damage on virions through attaching to the surface of viral
lipid envelope, followed by making a gradual leakage, which finally leading to breakage of the envelop and virus
inactivation. The in vivo test in HSV-1-infected mice showed that 8 (50 mg/kg/dose) possessed definite therapeutical
effect in the development of lesion score 46.
3.7. Anticancer activity
The anticancer properties of both extracts and isolated compounds of B. pandurata have been assessed in various
cancer cells. The results appear consistent with the traditional use of B. pandurata as a natural anti-cancer.
The methanol extract of B. pandurata was found to suppress tumor promotion through inhibition test of tumor
promoters-induced Esptein-Barr virus (EBV) activation in human B-lymphoblastoid (Raji) cells. The 20 g/mL of
extract strongly inhibited the teleocidin (20 ng)-induced EBV activation with inhibitory effect (IE) value of 70%
and was highly toxic according to cell viability value (CV30%). The expected active compound, 1, was found to
completely inhibit the EBV activation at a concentration of 25 M with high cell viability (CV>90%). The activity
of 1 (IC50 = 3.1 M) was comparable with that of a highly potent EBV activation inhibitor, 1-acetoxychavicol
acetate (ACA, IC50 = 1.5 M). Thus, the compound 1 is evaluated to be one of the most potent chalcone-type
inhibitors against EBV activation, as proposed by the authors 47. In other cancer cell models, the treatment with diet
containing 4% ethanolic extracts of B. pandurata in the azoxymethane (AOM)-induced aberrant crypt foci (ACF,
colon cancer) model in rat showed the reduction in the formation of ACFs although the difference was not
significant 13. The chloroform extract of B. pandurata also showed cytotoxic activity against human promyelocytic
(HL-60) cancer cells 48 and 100% preferential cytotoxicity (PC100) against human pancreatic cancer (PANC-1) cells
at 10 g/mL 17
Several pure compounds from B. pandurata have been tested for their anticancer activities. By using Ames test
system, compounds 1, 2, 8, 9, 24, and 28 were found to possess inhibitory activity against mutagenesis in Trp-P-1-
treated Salmonella typhimurium, with IC50 values of 5.9, 5.2, 5.3, 6.9, 12.1, and 12.7 M, respectively, and also
similar activity in Trp-P-2, PhIP- treated Salmonella typhimurium. The authors proposed that the antimutagenic
effect of these compounds is mainly based on inhibition of N-hydroxylation of Trp-P-2 since all of them showed
strong inhibitory activity against this reaction 15.
Compound 24 showed cytotoxicity in various cancer cells at low concentration such as in human colon cancer
(HT-29) cells with IC50 of 6.56 g/mL 13,49, androgen-independent human prostate cancer (PC3 and DU145) cells
with IC50 of 13.5 - 14 M 50, human breast cancer (MCF-7) cells with IC50 of 3.75 g/mL 13, human pancreatic
cancer (PANC-1) cells with PC100 of 10 g/mL, and human non-small cell lung cancer (A549) cells with IC50 of 4.4
g/mL 51. The cytotoxic effects of 24 were found to be accompanied by inducing of apoptosis these cancer cells.
Yun and co-workers found that at apoptosis-inducing concentration, 24 caused cleavage of poli(ADP-ribose)
polymerase (PARP) with a concomitant decrease in procaspase-3 protein in HT-29 cancer cells 49. This result was
strongly supported by Kirana and co-workers who found that in 24-treated HT-29 cancer cells, there were alterations
in the distribution of DNA content where the proportion of cells in the G0/G1 phase increased more significant than
in untreated cells, whereas in the S phase as well as in the G2/M phase was slightly reduced. In conclusion, 24
arrested cancer cells in the G0/G1 phase. In addition, the 24-treated HT-29 cells showed some features of apoptosis
such as membrane blebbing, chromatin condensation and/or nuclear fragmentation and apoptotic bodies 13. Also, in
both 24-treated PC3 and DU145 cells, 24 dose-dependently caused the cleavage of native 116 kDa PARP protein
into its characteristic 85 kDA protein and degradation of acinus. The mitochondrial-dependent apoptotic pathway
was found to be involved in the mechanism of apoptosis caused by 24 which shown with a decrease of expression of
initiator caspases -9 and -8 as well as procaspases -6 and -3 and the increase of ratio Bax:Bcl-2. Treatment of 24 in
both cell types also enhanced the expression of both Fas receptor protein, an apoptosis-inducing death receptor, and
TNF-related apoptosis-inducing ligand (TRAIL) protein. In the cell cycle regulation, unlike in 24-treated HT-29
cells, 24 was found to dose-dependently arrest the cell cycle in the G2/M phase in both cell types through reducing
the protein level of cyclin B1, cdc25C and cdc2, the protein members that regulate cell proliferation from G2 to M
phase, and up-regulating the expression of cdk inhibitors p21WAF/Ci1 and p27Kip1, leading then to cell growth
inhibition and apoptotic death 50. In A549 cells, 24 induced apoptosis through inhibition of NF-B translocation
from cytoplasm to nuclei and arrested cell in G2/M phase 51.
By using similar cancer cell, A549 cells, compound 22 was also comprehensively studied for its apoptotic activity
and mechanism in this treated cancer cell. 22 exhibited cytotoxic effect in A549 cells with an IC50 of 20 g/mL. At
apoptosis-inducing concentration (20 to 50 g/mL), 22 caused alterations in nuclear intensity and increase in
permeability of cells. 22 significantly reduced the mitochondrial membrane potential (MMP) on cells and then
triggered the cells to translocate the cytochrome c from mitochondria into cytosol during apoptosis. 22 was also
found to induce apoptosis through both intrinsic and extrinsic pathway by stimulating the expression of caspase-9
and caspases -3, -6, and -7 and increasing the ratio Bax:Bcl-2. The cell cycle analysis showed that 22 arrested cells
in sub-G1 phase 52
Other compounds were also tested for their anticancer activities. In PANC-1 cancer cells, compound 33 together
with 24 showed the highest cytotoxicity with PC100 value of 2.5 M, while the other compounds showed activities as
follows: 27 and 51 (PC100 = 8 M); 26, 30, 31, 35, and 72 (PC100 = 16 M); 9, 22, 23, 32, and 50 (PC100 = 64 M);
48 and 49 (PC100 = 128 M); and 1, 5, 9, 11, 9, 13, 14, 29, and 71 (PC100 > 256 M). As comparison, taxol did not
show cytotoxicity at concentration test (PC100 > 256 M) and arctigenin had PC100 value of 1 M 17. In addition, the
new panduratin derivatives, compounds 42, 43, 45, 46, 74, and 75, showed mild activity with PC100 values of 128
M, while compound 44, showed weak activity with PC100 value of > 256 M 8. These results showed that
methoxyl moiety at C-4 and hydroxyl moiety at C-2 and C-6 (in 24 and 33) are important for high activity, and any
modification, the presence of additional ring (in 42 46), and the absence of a substituted aryl group (in 74 and 75)
decrease activity, as proposed by the authors 8,17. In addition, twenty one compounds showed inhibitory activity on
aminopeptidase N (APN), a Zn+ -dependent metalloprotease that plays an important role in tumor-cell invasion,
extracellular matrix degradation, angiogenesis and tumor metastasis. All compounds were found to significantly
inhibit APN activity at 10 to 30 M. At concentration of 30 M, compounds 24, 28, 34, 37, 40, 41, and 72 showed
potent inhibitory activity (>50% inhibition), followed by compounds 11, 14, 16, 35, 36, 38, and 39 (20 to 40%
inhibition), and compounds 8, 9, 13, 15, and 18 (10 to 18% inhibition) 23.
3.8. Anti-aging activity
Compounds 24 and 28 as well as the extract of B. pandurata exhibited protective effect as anti-aging in human
skin fibroblast cells against ultraviolet (UV) radiation. The UV light induces photoaging by up-regulating MMP
activities and decreasing collagen synthesis. Interestingly, 24, in the range of 0.001 to 0.1 M, could reduce the
effect of UV radiation in fibroblast cells by reducing the expression of MMP-1 and elevating the expression of type-
1 procollagen 53. Compound 24 and 28 were also found to significantly reduce the UV-induced MMP-1 expression
through deactivation of MAPKs signaling molecules (ERK, p38 and JNK) resulted in the decrease of expression c-
Fos and c-Jun phosphorylation, which, in turn, led to inhibition of AP-1 DNA binding activity 54,55. The extract of B.
pandurata also showed strong inhibitory activity as anti-aging at 0.01 to 0.5 g/mL with the similar mechanism 56.
These results showed that compounds 24 and 28 as well as the extract have potent as new natural anti-aging agents,
as proposed by the authors.
3.9. Inhibitory effect in melanogenesis biosynthesis and tryrosinase activity
Compounds 24, 26, and 28 were found to possess strong activity in inhibiting of melanogenesis. The skin
pigmentation depends on melanin synthesis and the high accumulation of melanin can lead to the
hyperpigmentation. Interestingly, compared with known depigmenting agents, compounds 24, 26, and 28 inhibited
the melanogenesis in murine melan-a melanocytes more significant with IC50 values of 9.6, 10.64, and 23.25 M,
respectively. All of them also significantly inhibited the tyrosinase activity, an enzyme that catalyzes the
hydroxylation of tyrosine to dihydroxyphenilalanine (DOPA) leading to the accumulation of melanin as a
endproduct, with IC50 values of 8.2, 10.5, and >30 M, respectively. In addition, they were able to reduce the
expression on tyrosinase proteins 57,58. It seems, because having antioxidant properties, they probably prevent the
oxidation step in melanin biosynthesis.
3.10. Antiulcer activity
The methanol extract of B. pandurata and its bioactive compound, 8, were tested for their in vivo antiulcer
activity. They showed protective effect on in vivo gastric ulceration induced by ethanol in rats by increasing the
gastric mucus content, reducing area of gastric ulcer formation, and inhibiting of leucocytes infiltration of gastric
wall 59
3.11. Anti-parasitic activity
The extract of B. pandurata showed potent activity against protozoan parasites of humans. Both chloroform and
methanol extract of B. pandurata exhibited in vitro anti-giardial activity on Giardia intestinalis, a protozoan parasite
that causes some acute infections such as chronic diarrhoea in HIV/AIDS patients, with IC50 values of 44.48 and
78.30 g/mL 60. These extracts also showed anti-amoebic activity on Entamoeba hitolytica with IC50 values of 45.8
and 57.6 g/mL 61. However, in vivo application of B. pandurata powder in mouse pellets as a diet did not show any
effect on reducing inflammatory in Opisthorchis viverri-infected liver cells 62.
3.12. Anti obesity
Compound 24 and the ethanol extract of B. pandurata were tested their potent anti-obesity activity in 3T3-L1
murine adipocytes, HepG2 human liver carcinoma cells, L6 rat skeletal muscle cells and HeLa cervical carcinoma
cells 63,64. Compound 24 was found to regulate lipid metabolism by activating the LKB1-dependent AMPK (AMP-
activated protein kinase) signaling by which 24 increased phosphorylation of the AMPK catalytic subunit and
stimulated AMPK-dependent phosphorylation of acetyl-CoA carboxylase (ACC) and p38 AMPK, down-regulated
the expression of lipid synthesis proteins and up-regulated the expression of genes involved in fatty acid oxidation,
increased one of the MAPK activators (ratio AMP/ATP) by decreasing the cytosolic ATP levels, and increased
LKBI phosphorylation 63. The extract of B. pandurata also revealed similar molecular mechanism in which the
extract treatment in MDI-treated 3T3-L1 and insulin-induced HePG2 cells decreased triglyceride accumulation in
both cells by activating AMPK signaling and regulating the expression of lipid metabolism-related proteins 64
Compound 24 as well as the extract were found to attenuate high-fat diet (HFD)-induced obesity and fatty liver in
mice by activating KBI-dependent AMPK signaling. Both oral administration of 24 (50 mg/kg/day) and extract (200
mg/kg/day) significantly decreased weight gain and fat mass without reducing food intake. They also decreased
serum levels of cholesterol and triglyceride, decreased fat content in the liver and the muscle, and activated of
LKB1-dependent AMPK signaling 63,64.
4. Biotechnological Approach to produce biological active compounds of B. pandurata
According to the composition of secondary metabolites, B. pandurata contains a large number of flavonoids and
more than 60% of them are prenylated flavonoids (Table 1). Most of them, particularly prenylated flavonoids,
exhibited wide range pharmacological effect with high activity. SAR analysis revealed that flavonoids containing
geranyl moiety extremely determined for high biological activity. The presence of lipophilic prenyl group is
supposed to help enhance their transmembrane transport and interaction with the target proteins 65,66. Since the wide
variety of their biological effect, few of them have been developing as active compounds in many commercial
products such as drug, food and body care products. Panduratin derivatives (24, 26, and 28) and B. pandurata extract
have been patented by Hwang and co-workers for their usage as active ingredients in an anti-halitosis formula for
safe application to the buccal cavity without side effects 67. Additionally, they also patented all of them as effective
ingredients in cosmetics as anti-aging, and for food and pharmaceutical composition 68. Compound 24, due to its
acne induced suppression, antibacterial as well as anti-inflammatory effect, has been developing by an Koreans
company for its application in many skin care products such as lotions, creams, packs, ointment, etc 69. These facts,
however, are strongly believed leading to the high demand of these compounds, which, in turn, the supply of them
might not meet the world demand due to their low content in plant resources and the limiting production capacity of
this plant resources itself.
Attempts to produce such valuable compounds from this plant have been done through chemical synthesize and
plant cell/tissue culture. Long before panduratin derivatives were synthesized, compounds 22 and 23 are the first
compounds which were successfully chemically synthesized through acid-catalyzed 14,21. Various methods have
been shown to successfully synthesize Diels-Alder compounds from B. pandurata, i.e., compounds 24, 28, 33, 36,
74, and 75 70-72. Compound 24 and 36 were successfully synthesized in four steps through Diels-Alder cycloaddition.
Under moderate temperature reaction (100-150oC) and no catalyst, both of them were produced as a mixture in 89%
yield 70. By using silica-supported silver nanoparticle as a catalyst, over 70% yield of 24 was obtained via Diels-
Alder cycloaddition of 2-hydroxychalcone and trans--ocimene 71. Interestingly, cycloaddition method developed
by Pasfield et al. 72 by using high pressure Diels-Alder reaction was able to synthesize compound 74 and 75 via
cycloaddition of cinnamic acid and (E)-ocimene, which, then, further reaction produced compound 24 and 28 in five
steps. Compound 33 and 2-hydroxypanduratin A were also obtained by changing cinnamic acid with
cinnamaldehyde as a dienophile.
Some researchers successfully developed the plant tissue and cell culture of B. pandurata, either for rapid
micropropagation or for secondary metabolite production. The in vitro rapid regeneration of B. pandurata has been
well achieved via somatic embyrogenesis 73, shoot-derived calli 74, and shoot bud explants 75. All of regenerated
plantlets were successfully acclimatized in soil. Interestingly, the cell suspension culture of B. pandurata was found
to enable to produce some of the unprenylated and prenylated flavonoids 76,77. In cell suspension culture fed with
phenylalanine precursor, the level production of 8, 9, 11, 24, and 28 were significantly elevated 76, whereas abiotic
stress treatment such as temperature and agitation was also found to be able to affect level production of 1, 8, 9, 11,
and 24 in cell suspension culture of B. pandurata 77. These results reveal us that the chance to produce some of the
valuable compounds is highly possible through in vitro culture.
Table 1 Summary of secondary metabolites from rhizome of B. pandurata

Compounds References Compounds References
Chalcones: Prenylated Chalcones
Cardamonin (1) Jaipetch et al. (1982) Boesenbergin A (22) Jaipetch et al. (1982)
Pinocembrin chalcone (2) Trakoontivakorn et al. (2001) Boesenbergin B (23) Mahidol et al. (1984)
Helichrysetin (3) Cheenpracha et al. (2006) (+)-panduratin A (24) Tuntiwachwuttikul et al.
(1984)
2,6-dihydroxy-4- Jaipetch et al. (1982) Rubranine (25) Tuntiwachwuttikul et al.
methoxychalcone (4) (1984)
Flavokawain C (5) Win et al. (2007) (-)-isopanduratin A2 (26) Pandji et al. (1993)
2,4,6-trihydroxychalcone (6) Tewtrakul et al. (2009) ()-isopanduratin A1 (27) Pandji et al. (1993)
Uvangoletin (7) Tewtrakul et al. (2009) (+)-4-hydroxypanduratin A (28) Trakoontivakorn et al. (2001)
Flavanones: ()-panduratin C (29) Cheenpracha et al. (2006)
Pinostrobin (8) Jaipetch et al. (1982) 2,4-dihydroxy-3-(1-geranyl)- Win et al. (2007)
6-methoxychalcone (30)
Pinocembrin (9) Jaipetch et al. (1982) (1R,2S,6S)-2- Win et al. (2007)
hydroxyisopanduratin A (31)
5,7-dimethoxyflavanone (10) Jaipetch et al. (1983) ()-6-methoxypanduratin A (32) Win et al. (2007)
Alpinetin (11) Pandji et al. (1993) (-)-nicolaioidesin B (33) Win et al. (2007)
Sakuranetin (12) Tuchinda et al. (2002) (-)-panduratin A (34) Morikawa et al. (2008)
7,4-dihydroxy-5- Win et al. (2007) (-)-4-hydroxypanduratin A (35) Morikawa et al. (2008)
methoxyflavanone (13)
Flavones: (+)-isopanduratin A (36) Morikawa et al. (2008)
Tectochrysin (14) Jaipetch et al. (1983) (-)-isopanduratin A (37) Morikawa et al. (2008)
5,7-dimethoxyflavone (15) Jaipetch et al. (1983) (+)-krachaizin A (38) Morikawa et al. (2008)
5-hydroxy-3,7-dimethoxyflavone Jaipetch et al. (1983) (-)-krachaizin A (39) Morikawa et al. (2008)
(16)
5,7,4-trimethoxyflavone (17) Jaipetch et al. (1983) (+)-krachaizin B (40) Morikawa et al. (2008)
5-hydroxy-7,4- Jaipetch et al. (1983) (-)-krachaizin B (41) Morikawa et al. (2008)
dimethoxyflavone (18)
5,7,3,4-tetramethoxyflavone Jaipetch et al. (1983) Panduratin D (42) Win et al. (2008)
(19)
5-hydroxy-3,7,4- Jaipetch et al. (1983) Panduratin E (43) Win et al. (2008)
trimethoxyflavone (20)
5-hydroxy-3,7,3,4- Jaipetch et al. (1983) Panduratin F (44) Win et al. (2008)
tetramethoxyflavone (21)
Prenylated Flavanones: Panduratin G(45) Win et al. (2008)
(2R)-8-geranylpinostrobin (48) Win et al. (2007) Panduratin B1 (46) Pancharoen et al. (1987)
(2S)-6-geranylpinostrobin (49) Win et al. (2007) Panduratin B2 (47) Pancharoen et al. (1987)
(2S)-7,8-dihydro-5-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-8-phenyl-2H,6H-benzo[1,2-b:5,4- Win et al. (2007)
b]dipyran-6-one (50)
(-)-6-geranylpinocembrin (51) Win et al. (2007) Other Compounds:

Rotundaflavone Ia (52) Morikawa et al. (2008) Dihydro-5,6-dehydrokawain Tuchinda et al. (2002)
(70)
Rotundaflavone Ib (53) Morikawa et al. (2008) 5,6-dehydrokawain (71) Win et al. (2007)
Rotundaflavone IIa (54) Morikawa et al. (2008) Geranyl-2,4-dihydroxy-6- Win et al. (2007)
phenethylbenzoate (72)
Rotundaflavone IIb (55) Morikawa et al. (2008) 2,4-dihydroxy-6- Morikawa et al. (2008)
phenethylbenzoic acid methyl
ester (73)
5,7-dihydroxy-8- Morikawa et al. (2008) Panduratin H (74) Win et al. (2008)
geranylflavanone (56)
7-methoxy-5-hydroxy-8- Morikawa et al. (2008) Panduratin I (75) Win et al. (2008)
geranylflavanone (57)
Essential oils (EOs): Pandji et al. (1993), bin Jantan
-terpinene (58), geraniol (59), camphor (60), -ocimene (61), 1,8-cineole (62), myrcene (63), borneol et al. (2001), Norajit et al.
(64), camphene (65), methyl cinnamate (66), terpineol (67), geranial (68), neral (69), citral, limonene, (2007), Miksusanti et al.
and 11-dodecen-1-ol (2008)
As one of many plants that produces prenylated flavonoids, the ability of B. pandurata to produce prenylated
flavonoids is also due to the presence of prenyltransferase (PTase). PTase is a key enzyme that catalyzes the
prenylation of plant phenolic compounds such as phenylpropanoids, flavonoids, xanthones, and coumarins, through
the addition of isoprene units, such as dimetylalyl (C5), geranyl (C10), or farnesyl (C15) into their main structure.
Many plants are known to produce prenylated flavonoids. However, only a few of PTases have been characterized
from plants i.e., kaempferol 8-dimethylallyl transferase from Epimedium diphyllum 78, naringenin 8-
dimethylallyltransferase 79, naringenin 8-prenyltransferase (SfN8DT-1), genistein 6-prenyltransferase (SfG6DT-1),
and isoliquiritigenin dimethylallyltransferase (SLDT) from Sophora flavescens 80,81, chalcone
dimethylallyltransferase from Morus nigra 82, pterocarpan 4-dimethylallyltransferase (G4DT) from Glycine max 83
and isoavonoid prenyltransferase gene (LaPT1) from white Lupinus albus 66.
Although some prenylated flavonoids have been successfully isolated from B. pandurata, the biosynthetic
pathway, PTase and genes encoding PTase in this plant have not yet been elucidated. According to its variety of
prenylated compounds, we strongly suppose that B. pandurata might express many specific PTases that catalyze
many different prenyl transfer reaction. If we assume that each given compound is catalyzed by specific PTase, so
there will be a few of specific PTases that involve in prenylation of phenylpropanoids, chalcones, flavanones, and
phenylbenzoic acid. Prenylation of phenylpropanoids (74) and panduratin-related compounds (24, 43, 46, etc) might
be catalyzed by one specific PTase that specifically transfers geranyl moiety as a prenyl donor into non aromatic
carbon position and then leads to rearrangement of geranyl to form cyclohexenyl moiety (Diels-Alder
cycloaddition). Different pattern of cyclohexenyl moiety in compound 38 might also be catalyzed by different
PTase. So far, there is no report about this kind of PTase that has been characterized from plants.
Other types of PTase are, probably, aromatic PTases that able to prenylate both of chalcones and flavanones,
specifically at, in chalcone, C-3 or C-5 aromatic and in flavanone, C-6 or C-8 aromatic, and this prenylation only
occurs in B ring not in A ring, since none of flavonoids with prenyl moiety in A ring has been reported from B.
pandurata. Interestingly, unlike several isolated PTases that specifically transfer dimethylallyl moiety 66,78-80,82,83,
most of PTases from B. pandurata are, perhaps, supposed as specific geranyltransferases due to the most of
prenylated products are geranylated flavonoids, except a prenylation occurred in B ring of compound 43 might be
catalyzed by a specific dimethylallyltransferase. In addition, we strongly supposed that the prenylation occurred in
both chalcones and flavanones is probably not catalyzed by one kind of PTase but by a series of PTases. It is
strongly supported that a chalcone dimethylallyltransferase from Morus nigra did not catalyze prenylation in
benzaldehyde, flavanones, and flavones but only accepted chalcones and an isoflavone genistein, due to its structural
similarity with chalcone, as prenyl acceptors 82. Also, a specific flavanone PTase from Sophora flavescens,
naringenin 8-prenyltransferase (SfN8DT-1), could only catalyze flavanone groups and did not accept chalcones,
flavones, isoflavones, and flavonols as prenyl acceptors 80.
Another possibility of PTase is geranyl transferase that is possibly involved in prenylation of compound 72.
Perhaps, this proposed PTase is belongs to PTases classes which is involved in ubiquinone and shikonin
biosynthesis. Interestingly, this PTase seems to catalyze O-prenylation in compound 72 which mainly occurred at
benzoyl position, unlike LePGT (4-hydroxybenzoate geranyltransferase), a key enzyme in shikonin biosynthesis,
isolated from Lithospermum erythrorhizon 84 and OsPPT1 (4-hydroxybenzoate polyprenyltransferase), a key enzyme
in ubiquinone biosynthesis, isolated from Oryza sativa 85, in which, both of them are only responsible for C-
prenylation in 4-hydroxybenzoate not for O-prenylation. Other isolated PTases that are possibly closely related to
this proposed PTase, accordingly to its pattern of prenylation, are Fnq26, a CloQ/Orf2 class of PTase, isolated from
Streptomyces cinnamonensis that catalyzed O-prenylation in both dihydroxynaphthalene and 4-hydroxybenzoate 86
and bergaptol 5-O-geranyltransferase (B5OGT), isolated from Citrus limon, that also catalyzed O-prenylation in
coumarins 87. However, our proposed PTase involved in prenylation of 72 might be a new class of PTase that could
be able to prenylate benzoic acid derivatives at carboxyl position to form benzoic acid prenyl ester derivatives,
unlike with Fnq26 that only prenylated at hydroxyl position 86 and LePGT 84 or OsPPT1 85 that only catalyzed C-
prenylation in 4-hydroxybenzoate.
Definitely, the generating of plant prenylated flavonoids involves three major metabolic pathways, i.e., shikimic
(phenylpropanoid) pathway, acetate/malonate (polyketide) pathway, and isoprenoid pathway. As shown its
phytochemistry profiles, we propose the biosynthetic schema of prenylated flavonoids that possibly occurs in B.
pandurata (Fig. 9). Eleven protein enzymes related to this pathway have been identified in phenylalanine-fed cell
culture of B. pandurata through proteomic analysis. These protein consist of hypothetical protein (HPosJ), involved
in oxidative reaction; cafeoyl-CoA-O-methyltransferase (COM), involved in feruloyl-CoA formation; dihydrolipoyl
dehydrogenase (DD), fructose biphosphate aldolase (FBP), pyruvate kinase (PK), pyruvate dehydrogenase (PD) and
aconitate hydratase/aconitase (ACO) which, all of them, are involved in glycolysis pathway and citrate cycle,
leading to the availability of energy and isoprenoid precursors such as acetyl-CoA, pyruvate, and D-glyceraldehyde-
3-phosphate; 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXPR), involved in 2-C-methyl-erythritol-4-
phosphate (MEP) formation; 1-hydroxy-2-methyl-butenyl-4-diphospate reductase (HMDPR), involved in
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) formation; ferredoxin-nitrite reductase
(FNR) and glutamine synthetase (GS), involved in nitrogen metabolism and assimilation 76.
Finally, we strongly suggest that a comprehensive research toward the biosynthetic of such valuable compounds
in B. pandurata should be carried out in order to help us to have a better understanding about prenylflavonoid
pathways, their regulation in plant, and possibility for biotechnological modification, in order to increase level of
prenylated flavonoid compounds production, especially plants that produce interesting pharmaceutical compounds.
The characterization of PTase enables us to synthesize valuable prenylated flavonoids and other prenylated
polyphenols through metabolic engineering. This enzyme also represents an interesting tool for the organic synthesis
of aromatic compounds since its ability as biocatalyst in the formation of C-C bonds. PTase is expected to help us in
the synthesis of prenylated aromatic compounds possessing important pharmacological activities, while in the
chemical synthesis it is difficult to be achieved in good yield.
Fig. 9 Proposed biosynthetic pathway of prenylated flavonoids in B. Pandurata via shikymic pathway and isoprenyl come from Isoprenoid
pathway
5. Concluding remarks and future perspectives
B. pandurata is one of the plants belonging to the genus of Zingiberaceae that has been traditionally used in folk
medicine for centuries. Interestingly, according to the phytochemical analysis, we found that not only essential oils that
commonly exist in gingers plant but many specific flavonoid compounds were found in rhizome of B. pandurata. More
than 60% of these flavonoids are new prenylated flavonoids that specifically exist only in this plant.
Through in vitro and in vivo assays toward both extract and isolated compounds of B. pandurata, the
ethnopharmacological potential of this plant was almost fully validated. The structure-activity relationships (SAR)
analysis of isolated compounds revealed that the potent biological activities of this plant, such as antioxidant 31, anti-
inflammatory 12, antibacterial 9,10,39-41, antiviral 16, and anticancer 17, was highly enhanced and relied on the presence of
prenyl subsituents that integrated into the main structure of flavonoids. That is, the prenyl substituents are therefore very
important for biological activities of flavonoids. This reason brought then researchers to finally make several attempts
in order to produce some valuable prenylated flavonoids via either chemical synthesize or plant cell culture. However,
plant cell culture is, we thought, the most valuable tool as an early step leading to the study of biosynthesis pathway
toward some precious prenylated flavonoids using metabolomic, genomic as well as proteomic study as already done by
Tan et al. 76. The specific PTases are thought to be involved in catalyzing prenylation on flavonoids, since many PTases
have been isolated from various plants that also contained prenylated flavonoids. Therefore, isolation and
characterization of PTases from B. pandurata is thought to be important due to their ability to catalyze prenyl transfer
reaction upon flavonoid, particularly the Diels-Alder cycloaddition reaction occurred in panduratin A and other
panduratin-related compounds that are very specific occurred in B. pandurata. This will eventually enable us to produce
prenylated compounds in desired amount by using transgenic plant as shown by Sasaki et al. 88 or genetically modified
microbe cultures as shown by Sugiyama et al. 89. In addition, PTases will probably help us as biocatalysts to catalyze
the prenylation of aromatic compounds and even in the formation of unnatural aromatic compounds as shown by Chen
et al. 90 that were able to prenylate a nonaromatic carbon of indolebutenone by using fungal indole prenyltransferase,
which, in turn, leading to generate an unnatural novel compound.
Acknowledgements
Part of this work was financially supported by the I-MHERE 2011 ITB.
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International Seminar on Natural Product Medicines, ISNPM 2012

Boesenbergia pandurata Roxb., An Indonesian Medicinal Plant:

Keywords: Boesenbergia pandurata, Essential oil, Prenylated flavonoid, Panduratin, Antibacterial

* Corresponding author. Tel.: +62-22-2504852 fax: +62-22-2504852

Fig. 1 Typical class of regular flavonoids in B. pandurata

2.1.1. Unprenylated flavonoids

Fig. 2 Regular chalcones in B. pandurata

Fig. 3 Regular flavanones in B. pandurata

namely 5,7,4-trimethoxyflavone (17), 5-hydroxy-7,4-dimethoxyflavone (18), 5,7,3,4-tetramethoxyflavone (19),

Fig. 4 Regular flavones in B. pandurata

2.1.2. Prenylated flavonoids

Fig. 5 Prenylated chalcones in B. pandurata

Fig. 6 Prenylated flavanones in B. pandurata

2.2. Essential oils (EOs)

Fig. 7 The major compounds of EOs in B. pandurata

2.3. Miscellaneous compounds

Fig. 8 Other compounds in B. pandurata

3.1. Antioxidant activity

3.3. Antifungal activity

3.4. Antibacterial activity

3.5. Anti-periodontal activity

3.6. Antiviral activity

3.7. Anticancer activity

3.8. Anti-aging activity

3.9. Inhibitory effect in melanogenesis biosynthesis and tryrosinase activity

3.10. Antiulcer activity

3.11. Anti-parasitic activity

3.12. Anti obesity

4. Biotechnological Approach to produce biological active compounds of B. pandurata

Table 1 Summary of secondary metabolites from rhizome of B. pandurata

(-)-6-geranylpinocembrin (51) Win et al. (2007) Other Compounds:

5. Concluding remarks and future perspectives

14. Jaipetch, T, Kanghae, S, Pancharoen, O, Patrick, V, Reutrakul, V, Tuntiwachwuttikul, P, White, A.Constituents

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