Bioscience, Biotechnology, and Biochemistry

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Production of Hydroxycitric Acid by

Microorganisms

Hiroyuki HIDA, Takashi YAMADA & Yasuhiro YAMADA

To cite this article: Hiroyuki HIDA, Takashi YAMADA & Yasuhiro YAMADA (2005) Production
of Hydroxycitric Acid by Microorganisms, Bioscience, Biotechnology, and Biochemistry, 69:8,
1555-1561, DOI: 10.1271/bbb.69.1555

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Biosci. Biotechnol. Biochem., 69 (8), 1555–1561, 2005
Production of Hydroxycitric Acid by Microorganisms
Hiroyuki H IDA, Takashi Y AMADA, and Yasuhiro Y AMADAy
Department of Applied Biological Science, Faculty of Life Science and Biotechnology, Fukuyama University,
Gakuen-cho, Fukuyama, Hiroshima 729-0212, Japan
Received March 7, 2005; Accepted May 18, 2005
Hydroxycitric acid (HCA) is a major acid component
of the tropical plants Garcinia cambogia and Hibiscus
subdariffa. (2S,3S)-HCA from G. cambogia was shown to
be a potent inhibitor of ATP citrate lyase (EC4.1.3.8),
which catalyzes the extramitochondrial cleavage of
citrate to oxaloacetate and acetyl-CoA. (2S,3R)-HCA
from H. subdariffa inhibits -amylase and -glucosidase,
leading to reduction of carbohydrate metabolism. The
availability of HCA is limited by the restricted habitat of
the plants as well as the difficulty of stereoselective
organic synthesis. Hence, we screened microorganisms
producing HCA to find an alternative source of optically
pure bulk HCA. Two strains, Streptomyces sp. U121 and
Bacillus megaterium G45C, were screened by HPLC
analysis. Particular metabolites were purified from their
culture broths and compared with authentic HCA from
plants. NMR studies indicated that the products are
identical to Hibiscus-type HCA. This is the first report
showing isolation of microorganisms producing HCA.
Key words: hydroxycitric acid; Garcinia cambogia; Hi-
biscus subdariffa; Streptomyces sp.; Bacil-
lus megaterium
Tropical plants are a rich source of valuable secon-
dary metabolites, and some of them are useful as
medicines and food additives. Hydroxycitric acid (HCA)
is a major acid component in the rinds of Garcinia
cambogia, which thrives prolifically on the Indian
subcontinent and in western Sri Lanka. The rinds are
used in combination with salt for curing fish in Southeast
Asian countries.1,2) HCA is also enriched in the calyxes
of Hibiscus subdariffa, which is cultivated in several
tropical and semitropical countries. The dried flowers
are used as roselle herb tea. Roselle juice is a popular
beverage and herbal medicine.3,4) Since HCA was
identified, its effects have been studied extensively
because of its curious bioactivities.5,6)
HCA contains two diastereomers forming two chiral
centers in their molecules. Each of these diastereomers
forms a lactone ring. Therefore there are eight stereo-
isomers including lactone forms and their pairs of
enantiomers (Fig. 1). G. cambogia and H. subdariffa
y
To whom correspondence should be addressed. Tel: +81-84-836-2112; Fax: +81-84-936-2457; E-mail: yamada@fubac.fukuyama-u.ac.jp
produce (2S,3S) and (2S,3R)-HCA respectively. The
former is a competitive inhibitor of ATP citrate lyase
(EC4.1.3.8), which catalyzes the extramitochondorial
fission of citrate to oxaloacetate and acetyl-CoA.5,7) The
subsequent decrease in acetyl-CoA represses fatty acid
synthesis and lipogenesis.8,9) Animal studies support
these effects, and have shown that (2S,3S)-HCA sup-
presses food intake,10) thereby inducing weight loss.11)
(2S,3R)-HCA has been shown to inhibit pancreatic
-
amylase and small intestine
-glucosidase.6,12) In a
human cell model system, it reduced carbohydrate
digestion.13) Hence natural HCA is considered a safe
diet supplement or a possible food additive for diabetes
treatment. Only certain species of plants are known to
produce HCA, and their growth is limited to tropical and
semitropical areas. These facts suggest that production
of natural HCA is restricted by plant distribution area
and climate. Furthermore, stereoselective organic syn-
thesis is difficult due to the presence of two asymmetric
carbons in the molecule. Hence an alternative method-
ology is required to supply large amounts of an optically
pure natural-type HCA economically.
In the present study, we attempted to isolate micro-
organisms that produce HCA. Screening was performed
by HPLC analysis using authentic HCA prepared from
G. cambogia and H. subdariffa as standard samples to
confirm their retention times. HCA purified from the
culture broth of isolated strains was identified by NMR
analysis. These results clearly indicated the production
of Hibiscus-type HCA by microorganisms.
Materials and Methods
Chemicals. Mannitol and citric acid were purchased
from Wako Pure Chemical Industries (Osaka, Japan).
trans-Aconitic acid was obtained from Tokyo Kasei
Kogyo (Tokyo, Japan). 2-Oxoglutaric acid and glucose
were purchased from Nacalai Tesque (Kyoto, Japan).
All other chemicals and solvents were of analytical
grade.
Synthesis of trans-epoxy aconitic acid. Trans-epoxy-
aconitic acid was prepared from trans-aconitic acid by
1556
Fig. 1. Structures of HCA Stereoisomers.
HCA has four stereoisomers, (2S,3S), (2R,3R), (2S,3R), and (2R,3S), and their lactone forms. G. cambogia and H. subdariffa produce (2S,3S)
and (2S,3R)-HCA, respectively.
oxidation with H2 O2 in the presence of a catalytic
amount of sodium tungstate in H2 O at pH 5.0 at
70
C.14) The yield was about 85%.
Purification of HCA from plant tissues. Authentic
(2S,3S)-HCA was purified as its methyl ester from dry
rinds of G. cambogia, which were a gift from Iwata
Chemical Co., Ltd. (Iwata, Japan). Dry rinds (10 g) were
stirred in 100 ml of methanol-sulfuric acid (10% v/v) for
2 h at 60
C. After it was cooled to room temperature,
the reaction mixture was diluted with saturated NaCl
solution (100 ml) and extracted three times with di-
chloromethane (100 ml). The dichloromethane layers
were combined and dried with anhydrous sodium
sulfate. Dichloromethane was evaporated after filtration.
The residue was purified by open column chromatog-
raphy using silica gel (10 g). Crude HCA methyl ester
dissolved in dichloromethane was adsorbed on silica gel
(1 g) and dried. The silica gel was suspended in a small
volume of n-hexane and placed in the top of a column
equilibrated with n-hexane. After it was washed with n-
hexane (100 ml), HCA methyl ester was eluted with
300 ml of n-hexane/ethyl acetate (1/1), and the elute
was fractionated into 5 ml fractions. All the fractions
were analyzed by thin-layer chromatography (TLC) on a
silica gel sheet (Silica gel 60 F254 , Merck, Darmstadt,
Germany) with a solvent system of n-hexane/ethyl
acetate (1/1). Fractions were combined and then con-
centrated. The purified product was analyzed and
identified by NMR. Authentic (2S,3R)-HCA was puri-
fied as a methyl ester from dried calyxes (5 g) of
H. subdariffa (Fukurodo Pharmacy, Tokyo). The puri-
fication procedure was the same as that described for
(2S,3S)-HCA from dry rinds of G. cambogia.
Isolation of microorganisms from nature. The main
sources of microorganisms were plants in botanical
H. HIDA et al.
gardens and soils in western Japan. Pieces of plant leaf,
fruit rind, and stem were placed directly on the surface
of agar plates containing a possible HCA precursor such
as citric acid (50 g), trans-aconitic acid (20 g), trans-
epoxyaconitic acid (20 g), mannitol (25 g) or Garcinia
extract (a gift from Iwata Chemical Co., Ltd.) (5 g) and
inorganic components (NaNO3 , 2 g; K2 HPO4 , 1 g;
MgSO4 , 1 g; KCl, 0.5 g; FeSO4 , 0.01 g per liter).
Alternatively, parts of plants and soil samples were
suspended in sterilized 0.9% NaCl. The supernatant was
diluted and spread on agar plates. Microorganisms from
several culture collections were also tested for HCA
production.
Screening of microorganisms producing HCA. An
isolated single colony was inoculated into 5 ml of
screening media in a 30-ml test tube containing (per
liter) 1 g of KH2 PO4 , 0.25 g of MgSO4 , 3 g of corn steep
liquor (Nihon Shokuhin Kako Tokyo) and a carbon
source such as 100 mM glucose, 10 mM citric acid,
10 mM 2-oxoglutaric acid, 10 mM trans-aconitic acid or
10 mM trans-epoxyaconitic acid. Test tubes were incu-
bated on a reciprocal shaker at 30
C for 48 h. The
culture broth was boiled for 10 min and centrifuged. The
supernatant was filtrated with a syringe filter unit
(Millex-LG 0.20 mm, Millipore, Bedford, MA) and
analyzed by HPLC (Model L-7100; Hitachi High
Technologies, Tokyo) at a flow rate of 1.0 ml/min on
a 10:7  300 mm GLC610 column (Hitachi) with 0.1%
H3 PO4 and a column temperature of 60
C. HCA and
other organic acids were detected by absorbance at
210 nm.
Purification of HCA from culture broth. Microorgan-
isms were cultured in 400 ml medium containing (per
liter) 1 g of KH2 PO4 , 0.25 g of MgSO4 , 3 g of corn steep
liquor and 100 mM glucose, using 2 liter-buffled conical
 Hydroxycitric Acid in Microorganisms 1557

1
flasks on a rotary shaker at 30 C for 72 h. The culture Table 1. H-NMR Data for HCA Methyl Ester from G. cambogia
broth (2-liter) was centrifuged (5000  g, JLA-10.500, Compound 1 (lactone) Compound 2 (nonlactone)
Beckman Coulter, Palo Alto, CA) to remove microbial Chemical shift Chemical shift
H J in Hz J in Hz
cells. Calcium carbonate (5 g) was added to the super- (ppm) (ppm)
natant to remove citrate by precipitation as a calcium Ha 2.79 d J ¼ 17:5 Hd 3.03–3.04 m
salt. The filtrate was treated with 10 ml of cation Hb 3.16 d J ¼ 17:5 He 3.03–3.04 m
exchanger (Amberlite IR120; Organo, Tokyo) to remove Hc 5.30 s Hf 4.91 s
calcium ions. Then, the solution was loaded onto a OCH3 3.60–3.90 s OCH3 3.60–3.90 s
column of Diaion HP-20 (100 ml, Mitsubishi Chemical Three CH3 O–protons belonging to the lactone and nonlactone forms
Co., Tokyo). The flow-through fraction was applied to appeared at 3.70–3.90 ppm.
column of anion exchanger resin (100 ml, Amberlite
IRA410JCL; Organo) and washed with 1 liter of H2 O. 1
Table 2. H-NMR Data for HCA Methyl Ester from H. subdariffa
The adsorbed acidic portion was eluted with 100 ml of
25% formic acid solution and concentrated. The residue Compound 3 (lactone) Compound 4 (nonlactone)
was dissolved in 100 ml of methanol-sulfuric acid (10% H
Chemical shift
J in Hz
Chemical shift
J in Hz
v/v) and stirred at 60
C for 30 min for esterification. (ppm) (ppm)
Purification of HCA methyl ester was performed as Ha 3.07 d J ¼ 17:5 Hd 2.97 d J ¼ 16:4
described in authentic plant HCA. At the final purifica- Hb 2.87 d J ¼ 17:5 He 3.17 d J ¼ 16:4
tion step of HCA methyl ester from Streptomyces sp. Hc 5.11 s Hf 4.36 s
OCH3 3.60–3.90 s OCH3 3.60–3.90 s
U121, a preparative HPLC system was utilized (Model
LC-908, Recycling Preparative HPLC, Japan Analytical Three CH3 O–protons belonging to the lactone and nonlactone forms
appeared at 3.70–3.90 ppm.
Industry, Osaka; flow rate, 5.0 ml/min; Column, 20 
250 mm Inertsil ODS, GL Science, Tokyo; Solvent
system, CH3 CN/H2 O (65/35)).

Spectrometric analysis. Structures of purified HCA

methyl esters were determined by 1 H-NMR. NMR
signals were recorded on a JNML-AL series AL300 for
300 MHz (Japan Electron Optics Laboratories, Tokyo).
The molecular masses of HCA and its methyl ester were
estimated in negative ion mode by ESI-MS (Mariner
,
Applied Biosystems, Foster City, CA).

Measurement of HCA concentration. The concentra-

tion of HCA was determined by HPLC analysis.
Calibration was linear at concentrations between 2 and
2,000 mg/liter with an r2 value of 0.999.

Results
Preparation of authentic HCA from G. cambogia and
H. subdariffa
Prior to isolation of microorganisms producing HCA,
we purified HCA from G. cambogia and H. subdariffa.
These HCA were used as authentic samples for screen-
ing by HPLC analysis and for identifying the microbial
products by NMR analysis. About 200 mg of (2S,3S) and
(2S,3R)-HCA methyl ester were obtained from 10 g of
dried rinds of Garcinia and 5 g of dried calyxes of
Hibiscus respectively.
The results of 1 H-NMR (in CDCl3 , 300 MHz) of
(2S,3S) and (2S,3R)-HCA methyl ester are shown in
Tables 1 and 2, and the structures are illustrated in
Fig. 2. The coupling constants of Ha and Hb were nearly
identical to those in the previous reports.12,15,16) More-
over, methanolysis of HCA lactone methyl ester in the
presence of a small amount of sodium methoxide led to
increased signals of Hd and He and decreased signals of
Fig. 2. Structures of HCA Methyl Esters and Their Lactones
Prepared from G. cambogia (1 and 2) and H. subdariffa (3 and 4).
Authentic HCA was purified as its methyl ester. ESI-MS data
indicated that they were a mixture of nonlactone HCA dimethyl
ester and lactone HCA monomethyl ester.
Ha and Hb (data not shown). These results suggest that
Hd and He belong to a nonlactone form, and that both
lactone and nonlactone forms exist in purified samples.
HCA methyl esters were subjected to hydrolysis with
NaOH. The 1 H-NMR spectrum (in D2 O, 300 MHz) of
(2S,3S)-HCA from G. cambogia was lactone form:
2.79 ppm (Ha, d, J ¼ 18 Hz), 3.14 ppm (Hb, d, J ¼
18 Hz), 5.30 ppm (Hc, s); nonlactone form: 2.91 ppm
(He, d, J ¼ 16:5 Hz) and 3.01 ppm (Hd, d, J ¼ 16:5 Hz),
4.90 ppm (Hf, s). That of (2S,3R)-HCA from H. sub-
dariffa was lactone form: 2.70 ppm (Hb, d, J ¼
1558
Fig. 3. HPLC Profiles of Extract of Dried Flowers of H. subdariffa (A) and Purified HCA (B).
A, HCA lactone and nonlactone are major acid components in the calyxes of H. subdariffa. B, Peaks 1 and 2 correspond to HCA lactone and
nonlactone respectively.
17:7 Hz), 3.24 ppm (Ha, d, J ¼ 17:7 Hz), 5.11 ppm (Hc,
s); nonlactone form: 2.84 and 2.97 ppm (He, Hd, d,
J ¼ 16:2 Hz), 4.37 ppm (Hf, s). The coupling constants
of Ha and Hb were consistent with those described
previously.12,16) On ESI-MS, both HCA methyl esters
gave ½M  H ions at m=z 203 and 235, indicating that
they included the lactone of monomethyl ester and the
nonlactone of dimethyl ester. HCA obtained by alkaline
hydrolysis showed ½M  H ions at m=z 189 and 207,
which correspond to those of HCA lactone and non-
lactone respectively.
(2S,3S) and (2S,3R)-HCA showed almost identical
retention times at 9.30 (peak 1) and 10.20 min (peak 2)
on an HPLC chromatogram (Fig. 3B). The HCA sample
present in the alkaline solution produced peak 2,
suggesting that peaks 1 and 2 correspond to lactone
and nonlactone forms respectively. Additionally, they
were detected in crude extracts of dried rinds of
G. cambogia (data not shown) and dried flowers of
H. subdariffa (Fig. 3A) as major acid components.
Screening microorganisms producing HCA
Isolation of microorganisms from various ecological
niches was carried out using agar plates containing
possible HCA precursors as a carbon source. About
2,000 strains were screened by HPLC analysis. There
were 30 strains generating two metabolites with reten-
tion times similar to those of HCA lactone and non-
lactone. Of these, two strains, U121 and G45C, which
H. HIDA et al.
were selected on agar plates with Garcinia extract,
showed apparently higher production of the metabolites
(Fig. 4). The products were purified, and finally 11 and
13 mg of the methyl esters were obtained respectively.
The 1 H-NMR spectrum (in CDCl3 , 300 MHz) of the
methylester of U121 exhibited signals at 2.98 and
3.16 ppm (J ¼ 16:4 Hz) and 4.36 ppm, identical to those
of the authentic nonlactone form of Hibiscus-type HCA
methyl ester diastereoisomer (Tables 2 and 3). That of
the methyl ester of G45C showed signals at 2.87 and
3.07 ppm (J ¼ 17:5 Hz) and 5.11 ppm, identical to those
of the lactone form of Hibiscus-type HCA methyl ester
(Tables 2 and 3). Furthermore, chemical shifts in the
nonlactone form were found as well, suggesting that the
purified product includes both lactone and nonlactone
forms. In the 1 H-NMR spectra of the U121 and G45C
products, there was no signal corresponding to the
methyl ester of Garcinia-type diastereoisomer HCA
(Table 3). These results indicate that strains U121 and
G45C produced pure HCA of Hibiscus-type diaster-
eomers.
Identification of isolated strains
The isolated strain U121 had substrate mycelium,
aerial mycelium, arthrospores in verticals, and chains of
spores. The other isolated strain, G45C, was gram-
positive, motile, and rod-shaped, and showed spore
formation, and catalase-positive and oxidase-negative
reactions. Phenotypic analysis and morphological char-
 Hydroxycitric Acid in Microorganisms 1559

Fig. 4. HPLC Profiles of Supernatants from the Culture Broth of Streptomyces sp. U121 (A) and Bacillus megaterium G45C (B).
Both strains produced various metabolites including citric acid (retention time, 11.40 min), oxaloacetic acid (12.20 min) and malic acid
(13.80 min). Peaks 3 and 4 had the same retention times as HCA lactone and nonlactone respectively.

Table 3. 1
H-NMR Data for Products 1 and 2
Discussion
Product 1 Product 2 In this report, we describe the isolation of micro-
 Chemical shift Chemical shift organisms producing HCA. Authentic HCA was pre-
H J in Hz J in Hz
(ppm) (ppm)
pared from G. cambogia and H. subdariffa, and micro-
Ha 3.07 d J ¼ 17:5 organisms were screened from nature by HPLC analysis.
Hb 2.87 d J ¼ 17:5 The product was purified from culture broth and
Hc 5.11 s
Hd 2.98 d J ¼ 16:4 2.97 d J ¼ 16:4
identified as the methylester of HCA by NMR analysis.
He 3.16 d J ¼ 16:4 3.17 d J ¼ 16:4 To our knowledge, this is the first report of production of
Hf 4.36 s 4.36 s HCA by microorganisms.
OCH3 3.60–3.90 s 3.60–3.90 s NMR and ESI-MS data for purified authentic samples

Products 1 and 2 were obtained from Streptomyces sp. U121 and Bacillus indicate that they included a nonlactone form of
megaterium G45C respectively. dimethyl ester and a lactone form of monomethyl ester.
The lactone form data were consistent with those of
previous studies.12,15) HCA prepared by hydrolysis of the
acteristics strongly suggested that the strains were purified esters showed retention times similar to major
Streptomyces and Bacillus megaterium respectively. acid components of the extracts of Garcinia rinds and
With these results, the isolated strains were tentatively Hibiscus calyxes (Fig. 3). These results strongly indicate
designated Streptomyces sp. U121 and Bacillus mega- that authentic HCA samples were successfully prepared.
terium G45C respectively. Among more than 2,000 microbial strains, Streptomyces
sp. U121 and Bacillus megaterium G45C generated
Growth curve and production of HCA metabolites showing retention times similar to authentic
The growth of Bacillus megaterium G45C and its HCA on HPLC (Fig. 4). We supposed that they were
HCA production were investigated. Production of HCA possible HCA producers. The products were purified
paralleled cell growth. The strain yielded HCA after 12 h from their culture broths, and NMR analysis verified
and reached a maximum level of 22 mg/l at about 44 h them as HCA (Table 3). From the culture broth of
(Fig. 5). Streptomyces sp. U121 showed slower growth Streptomyces sp. U121, only nonlactone form HCA
and HCA production (data not shown). The maximum methyl ester was obtained. In this case, we used a
production was 30 mg/l at about 48 h. preparative HPLC system for the final purification
process of HCA. Lactone-form HCA was probably
eliminated by this step. We isolated three strains that
1560
Fig. 5. Production Curve of HCA from Bacillus megaterium G45C.
Symbols: , bacterial growth; , HCA productivity of the supernatant.
showed metabolite with a retention time of 10.20 min on
the HPLC chromatogram, but NMR data indicated that
the products were not HCA (data not shown). Therefore,
selection of strains that produced both HCA lactone and
nonlactone forms of metabolites appeared to be critical
for efficient screening in this HPLC system. Our finding
of two HCA producers is not surprising, because the
HPLC system has higher sensitivity and resolution than
paper chromatography or TLC, which were used to
screen producers of particular organic acids. Effective
HPLC analysis combined with NMR data allowed the
identification of HCA produced by microorganisms.
The two microbial strains isolated in this study proved
to yield Hibiscus-type HCA (diastereoisomers). It has
been reported that Hibiscus HCA inhibited pancreatic
-
amylase and small intestine
-glucosidase.6,12) Admin-
istration of these enzyme inhibitors was shown to
repress increases in blood glucose and insulin levels.17)
Acarbose, an
-glucosidase inhibitor, is widely used as a
therapeutic agent for diabetes treatment.17–19) However,
it must be used carefully due to side effects. Hibiscus
HCA is expected to be a safe food additive for
prevention of diabetes and obesity.12) Microbial produc-
tion is an alternative source of optically pure Hibiscus-
type HCA. Isolation of microorganisms producing
Garcinia-type HCA is a future project. In our HPLC
screening, we obtained 28 additional strains which were
found to be possible HCA producers besides Strepto-
myces sp. U121 and Bacillus megaterium G45C. Their
production levels were estimated to be 2 to 10 mg/l for
HCA, and structural identification is on going. Thus we
cannot exclude the possibility that some of them may
produce Garcinia-type HCA. At present, the maximum
HCA production is 30 mg/l by Streptomyces sp. U121
and 22 mg/l by Bacillus megaterium G45C, as estimated
by HPLC. The yields were lower than those of other
organic acid fermentations, such as citric acid by
H. HIDA et al.
Aspergillus niger (30–74 g/l),20) tartaric acid by Gluco-
nobacter suboxydans (5–12 g/l)21) and abscisic acid by
Cercospora rosicola (120 mg/l).22) Medium optimiza-
tion and selective breeding using mutagen and genome
shuffling are promising methods to improve HCA
production. Construction of HCA hyperproducers is
under way in our laboratory.
The biosynthetic pathway of HCA in plants and
microorganisms remains unclear. Enhancement of HCA
production due to the addition of possible HCA
precursors such as TCA cycle intermediates and other
analogs to their culture broth was not observed. Resting
cell reaction with candidate HCA precursors did not
elicit HCA production either (data not shown). There are
several examples indicating that plants and microorgan-
isms generate common secondary metabolites. Higher
plants and fungi have different routes for the biosyn-
thesis of abscisic acid.23,24) Gibberellin biosynthesis in
Gibberella fujikuroi is different from that in higher
plants.25) On the other hand, Phaeosphaeria sp. L487
produces GA1 through a pathway similar to that in
higher plants.26) Based on observation of the chemical
structure of HCA, the biosynthetic pathway is probably
not complicated, but it must be carefully explored.
Construction of HCA hyperproducers might help to
establish its biosynthetic principles.
In conclusion, we discovered microorganisms that
produce HCA, providing a potential alternative source to
Hibiscus HCA.
Acknowledgments
We are grateful to Dr. H. Funahashi and M. Nakao in
Iwata Chemical Co., Ltd., for the gift of Garcinia extract
and their invaluable advice. We thank S. Goto and T.
Sambe for screening of microorganisms. We would like
to acknowledge the ESI-MS analysis of Dr. K. Tsurusaki
 Hydroxycitric Acid in Microorganisms
in the Department of Environment and Information
Sciences of Fukuyama University. This work was in part
supported by Iwata Chemical Co., Ltd., and ‘‘High-Tech
Research Center’’ Project for Private Universities on
Evolution of Green Science for Quality Improvement of
Environmental and Health: matching fund subsidy from
MEXT (Ministry of Education, Culture, Sports, Science
and Technology), 2004–2008.
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