Food Chemistry 120 (2010) 235–239

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Food Chemistry

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Hydroxycitric acid lactone and its salts: Preparation and appetitesuppression studies

G. Venkateswara Rao, A.C. Karunakara, R.R. Santhosh Babu, D. Ranjit, G. Chandrasekara Reddy *

Vittal Mallya Scientific Research Foundation, P.B. No. 406, K.R. Road, Bangalore 560004, Karnataka, India

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

Article history:Received 7 May 2009Received in revised form 28 August 2009Accepted 6 October 2009

Keywords:Hydroxycitric acid lactoneAppetite suppressionWeight reductionIn-vivo studies

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.10.014

* Corresponding author. Tel.: +91 80 26611664; faxE-mail address: gcreddy@vmsrf.org (G. Chandrasek

(�) Hydroxycitric acid lactone (HCAL) has been prepared in pure form (>98%) and converted into differentsalts of Group-IA and IIA metals with definite composition for the first time. The lactone is stable at roomtemperature but in aqueous solution it exists in equilibrium with its acid counterpart. An HPLC methodhas been developed to quantify (�) hydroxycitric acid (HCA) and its lactone. In-vivo studies, using malerats of the Wistar albino strain, revealed the interesting finding that HCAL exhibited better appetite sup-pression than did hydroxycitric acid. The continuous shift in equilibrium between HCA and HCAL due tothe depletion of HCA in animal systems (slow release mechanism) could be the possible reason for thisimproved activity.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

(�) Hydroxycitric acid (HCA) is a constituent of Garcinia cambo-gia, Garcinia indica and Garcinia atroviridis, which is widely used infood preparation as a soaring agent and is known to cause appetitesuppression (Conte, 1994; Heymsfield et al., 1998; Lewis & Neela-kantan, 1965; Thom, 1996). Watson and Lowenstein (1970), andWatson, Fang, and Lowenstein (1969) showed that HCA was a po-tent competitive inhibitor of the extra-mitochondrial enzymeadenosine triphosphate citrate (Pro-3S)-lyase. Subsequentlyin vitro and in-vivo studies revealed that HCA not only inhibitedthe action of citrate lyase and suppressed de novo fatty acid synthe-sis (Lowenstein, 1971) but also increased rates of hepatic glycogensynthesis (Sullivan, Triscari, & Neal Miller, 1974), suppressed foodintake (Sullivan, Triscari, Hamilton, & Neal Miller, 1973) and de-creased body weight gain (Nageswara Rao & Sakeriak, 1988). Re-cently it has been established that a high dose of HCA waseffective in suppressing fat accumulation in developing male Zuc-ker obese rats but was found to be highly toxic to the testis (Hay-amizu et al., 2003; Saito et al., 2005). Various salts of this naturallyoccurring HCA are now being used in nutraceuticals and incorpo-rated into many preparations in weight management and are soldover-the-counter to consumers all over the world (Hobbs, 1994).

HCA is 1,2-dihydroxy propane 1,2,3-tricarboxylic acid which isa c-hydroxy acid and gets easily lactonised to form (2S,3S)-tetra-hydro-3-hydroxy-5-oxo-2,3-furandicarboxylic acid [HCAL (I)](Fig. 1). HCA preparations usually contain either a crude extract

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: +91 80 26612806.ara Reddy).

of Garcinia (Krishnamurthy, Ravindranath, & Sathyagalam, 1988)or its salts, such as a tripotassium salt solution (Lewis, 1967) andwater-soluble mixed double salts of Group-IA and IIA metals (Bal-asubramanyam, Chandrasekar, Romados, & Subba Rao, 2000). Itspreparation involves water extraction of Garcinia rind, followedby purification using ion-exchange resins and converting it intodifferent salts. Many of the HCA salts currently available are crudepreparations with no definite metal content. In the context of strin-gent regulations being enforced on the nutraceuticals with respectto quality and reproducibility, it is relevant to investigate newmethodologies in order to make pure products with definite com-position. Efforts to make these salts with defined purity/composi-tion resulted in the isolation of pure crystalline HCAL.

HCAL was isolated in a pure crystalline form for the first timeand its properties, were extensively studied, including stabilityand crystal structure (Sudarshan, Srijita, Venkateswara Rao,Chandrasekara Reddy, & Guru Row, 2007). Pure HCAL thus pre-pared was used for making different salts of known composition(Venkateswara Rao, Karunakara, Manoj, Kush, & ChandrasekaraReddy, 2008). The appetite suppression activity of pure HCAL interms of feed intake and reduction in weight gain has been studiedin comparison with the pure HCA trisodium salt (IIa, Fig. 1).

2. Materials and methods

2.1. Materials

G. cambogia rind, and reference samples of hydroxycitric acidsalts, sodium hydroxide, potassium hydroxide, calcium carbonate,magnesium carbonate, ethyl acetate, acetone, and ethanol, were

O H

OH

OCOOH

COOH

CH2COO

CCOO

CHCOO

OH

OHY

X_

_

_

+

++

HCA(II)HCAL(I)

IIa = X, Y = Na

IIb = X, Y= K

IIc = X = K, Y = Ca

IId = X = Na, Y = Ca

IIe = X, Y = Mg

Fig. 1. Structures of (�) hydroxycitric acid lactone and its salts.

236 G. Venkateswara Rao et al. / Food Chemistry 120 (2010) 235–239

procured from local suppliers and used as obtained. HPLC gradeacetonitrile was purchased from Spectrochem of India and tetrabu-tyl ammonium sulphate (99% pure) was purchased from Sigma Al-drich, USA.

2.2. Instruments

Melting point was determined on an Acro melting point appara-tus and was uncorrected. 1H and 13C NMR spectra were recordedon a Bruker 200 MHz instrument. Chemical shift values (d) are gi-ven in parts per million downfield from the internal standard, tet-ramethylsilane. Mass spectrum was recorded using GCMS-QP2010S (Direct probe) and on a Q-TOF microTM AMPS MAX 10/6A system. Purity of HCA and its lactone were determined usingHPLC (Shimadzu Corporation, Japan). Optical rotations were mea-sured on a Jasco DIP-370 Digital Polarimeter (Japan spectrometricco. Ltd., Tokyo, Japan).

2.3. (2S,3S)-Tetrahydro-3-hydroxy-5-oxo-2,3-furandicarboxylic acid(I)

Dried G. cambogia fruit rinds (200 g) were cut into small piecesand soaked in water (250 ml) for 10 h. Aqueous extract was col-lected after squeezing and filtration into a separate vessel andthe process was twice repeated. The combined water extracts wereconcentrated to get a thick syrupy mass which was dissolved inacetone (250 ml) and the insoluble mass was filtered off. The ace-tone layer was evaporated to dryness and the thick mass obtainedwas reextracted into ethyl acetate (125 ml). The ethyl acetate layerwas treated with charcoal (4 g), filtered through charcoal, driedover anhydrous sodium sulphate and evaporated to get crude lac-tone. The crude material thus obtained was recrystallized by dis-solving in 70 ml of ethyl acetate, followed by the addition of35 ml of hexane to yield 25 g of crystalline HCAL, mp 176–178 �C; [a]D

25: +112.6� (c = 1.0 in ethyl acetate); HPLC purity>98%; 13C NMR (50 MHz, D2O) d 42.9, 76.8, 78.7, 178.2, 179.5,180.3; GC–MS (m/z): 190(M+).

Alternatively, compound I was made from commercially avail-able (�) hydroxycitric acid salts or its aqueous solution by neutral-izing with HCl/H2SO4 and evaporating the solution to dryness toget crude lactone. This lactone was purified by crystallization usingethyl acetate, hexane mixture.

2.4. Trisodium (2S,3S)-dihydroxy-1,2,3-propane tricarboxylate (IIa)

To an aqueous solution of I (950 g, 5 mol, in 5.0 l of water), wasadded 95% of required sodium hydroxide solution (600 g, 15 mol,in 2.5 l of water) and the whole heated to 60 �C for 2 h. The reactionmass was cooled to room temperature and adjusted to neutral pHusing the remaining quantity of sodium hydroxide solution. Theresultant solution was filtered and the material was precipitatedusing ethyl alcohol. The product, IIa, was filtered and dried undervacuum to give 1.3 kg (95%) of material. [a]D

25: �14.7� (c = 1.0 inwater); HPLC purity >99% (compared with reference sample).

2.5. Tripotassium (2S,3S)-dihydroxy 1,2,3-propane tricarboxylate (IIb)

To an aqueous solution of I (19.0 g, 100 mmol in 100 ml ofwater), was added 95% of required potassium hydroxide solution(19.0 g, 300 mmol in 5 ml of water) and the whole heated at60 �C for 2 h. The reaction mass was cooled to room temperatureand the pH adjusted to neutral using the remaining quantity ofpotassium hydroxide solution. The resultant solution was filteredand the material was precipitated using ethyl alcohol. The product,IIb, was filtered and dried under vacuum to give 30 g (93%) ofmaterial. [a]D

25: �12.2� (c = 1.0 in water); HPLC purity >99% (com-pared with reference sample).

2.6. Monopotassium, calcium (2S,3S)-dihydroxy 1,2,3-propanetricarboxylate (IIc)

To an aqueous solution of I (19.0 g, 100 mmol in 100 ml ofwater), was added calcium carbonate suspension (9.0 g, 90 mmol,in 5 ml of water) with stirring at room temperature for 2 h andthen 95% of required potassium hydroxide solution (5.5 g,119 mmol, in 25 ml of water) added. The solution was heated at60 �C for 2 more hours, cooled to room temperature and the pH ad-justed to neutral with remaining potassium hydroxide solution.The resultant solution was filtered and the material was precipi-tated using ethyl alcohol. The product, IIc, was filtered and driedunder vacuum to give 27.5 g (97%) of material. [a]D

25: �7.0�(c = 1.0 in water); HPLC purity >99% (compared with referencesample).

2.7. Monosodium, calcium (2S,3S)-dihydroxy 1,2,3-propanetricarboxylate (IId)

To an aqueous solution of I (19.0 g, 100 mmol in 100 ml ofwater), was added calcium carbonate suspension (9.0 g, 90 mmol,in 5 ml of water) with stirring at room temperature for 2 h andthen 95% of required sodium hydroxide solution (4.8 g, 120 mmol,in 25 ml of water) added. The solution was heated at 60 �C for 2more hours, cooled to room temperature and the pH adjusted toneutral with remaining sodium hydroxide solution. The resultantsolution was filtered and the material was precipitated using ethylalcohol. The product, IId, was filtered and dried under vacuum togive 26.0 g (97%) of material. [a]D

25: �25.6� (c = 1.0 in water); HPLCpurity >98% (compared with reference sample).

2.8. Magnesium (2S,3S)-dihydroxy 1,2,3-propane tricarboxylate (IIe)

To an aqueous solution of I (19.0 g, 100 mmol in 100 ml ofwater), was added 95% of required magnesium carbonate suspen-sion (10.8 g, 150 mmol, in 5 ml of water) with stirring at room tem-perature for 2 h and then the whole was heated to 60 �C for 2 morehours. The reaction mass was cooled to room temperature and thepH adjusted to neutral with the remaining quantity of magnesiumcarbonate suspension. The resultant solution was filtered and thematerial was precipitated using ethyl alcohol. The product, IIe,was filtered and dried under vacuum to give 23 g (97%) of material.[a]D

25: �7.6� (c = 1.0 in water); HPLC purity >99% (compared withreference sample).

2.9. Quantification of HCA and HCAL by HPLC

2.9.1. Preparation of standardStandard stock solutions of both HCA salts and HCAL were pre-

pared by dissolving accurately weighed amounts (about 10 mg) ofpure standards in 1 ml of milli-Q-water, and solutions were pre-served at 4 �C. Composite working standard solutions of concentra-tion 730, 365, 146, 109.5, 73, 36.5, 1.8 lg/ml of HCA and 980, 490,

CALIBRATION GRAPH OF HCA

y = 2666.2x - 7415.2

R 2 = 0.9998

-500000

0

500000 1000000 1500000 2000000 2500000

0 200 400 600 800

CONCENTRATION IN µg/ml

RE

SP

ON

SE

IN m

AU

CALIBRATION GRAPH OF HCAL

y = 1999.9x - 5603R 2 = 0.9999

-500000

0

500000

1000000

1500000

2000000

2500000

0 200 400 600 800 1000 1200

CONCENTRATION IN µg/ml

RE

SP

ON

SE

IN m

AU

Fig. 2. Linearity graphs for the HPLC method validation.

G. Venkateswara Rao et al. / Food Chemistry 120 (2010) 235–239 237

196, 147, 98, 49, 2.45 lg/ml of HCAL were prepared when requiredby diluting stock solutions with milli-Q-water.

2.9.2. Preparation of sampleThree sets of HCAL solutions were prepared by dissolving

100 mg of HCAL in 100 ml of milli-Q-water. These solutions werekept at 30, 50 and 80 �C by keeping in preheated water bathsand by maintaining temperature for about 6 h to study the stabilityof the lactone. Samples were withdrawn at 1 h intervals to checkthe HCAL conversion into HCA using HPLC.

2.9.3. HPLC–PDA conditionsAll analyses were carried out using Shimadzu HPLC equipment

composed of a binary gradient pump, a Rheodyne manual loopinjector with a 20 ll loop, column oven and a PDA detector. Sepa-ration of HCA and HCAL was achieved by using an Inertsil ODS-3VC18 column, 250 � 4.6 mm, 5 lm particle size, as stationary phaseand a mixture of 5 mM tetrabutyl ammonium sulphate:acetonitrile(98:2) as mobile phase. The eluent was monitored at wavelength210 nm at flow rate 1 ml/min at column oven temperature 30 �C.The data were processed with class-vp 6.12 software, taking intoaccount the peak areas of analytes.

2.9.4. Validation of HPLC methodThe linearity of the method was evaluated by analyzing series of

standard solutions of HCA and HCAL at concentrations of 1.8–730and 2.45–980 lg/ml, respectively, on HPLC (the retention timesof HCA and HCAL were found to be 7.3 and 24.0 min, respectively)(Fig. 2). Regression curves for HCA and HCAL were obtained byplotting concentration against peak area (average of 3 runs). Thelimit of detection (LOD), calculated as the amount of analyte re-quired to obtain a signal to noise ratio of 3 and limit of quantifica-tion (LOQ), that is, the lowest concentration to yield a signal tonoise ratio of 10. The method precision was evaluated by analyzingsamples in triplicate.

2.9.5. Optical rotation (a)Sample solutions were prepared by accurately weighing about

1 g of HCA salts/HCAL and dissolving in 100 ml of milli-Q-water.Optical rotations were measured at a wavelength of 589 nm andat 25 �C. Specific rotation [a]D was calculated using the formula:a� 100/L � c where a is optical rotation, L is cell length in decime-tres and c is concentration of the solution in grammes per 100 ml.The HCAL solutions, which were maintained at 30, 50, and 80 �C,were collected at regular intervals and were cooled to 25 �C beforemeasuring optical rotations.

2.10. Animal studies

Male rats of the Wistar albino strain weighing from 130–180 g,were maintained under hygienic conditions and kept on a standardrodent diet. A total of 42 rats were weighed, staggered and as-

signed to 7 groups of six animals each. Assignment of rats to 7groups was made in such a way that each group had mean and to-tal body weights similar to the other groups. All rats were housedindividually in pairs in metal mesh cages and with individual foodcups for weighed diets. They were housed in a light-controlledroom (12 h light, 6 am to 6 pm and 12 h dark cycle, 6 pm to 6am). All the rats were allowed free access to drinking water andfed ad labium prior to the start of the experiment. Animals weremaintained on a single 5 h meal per day (12–5 pm) for 7 days asan acclimatization period, followed by 24 h of fasting prior to start-ing the experiment. Test compounds were fed orally prior to thestart of the meal and then rats were fed from 12 to 5 pm daily.The experiment was conducted for eight weeks. Daily feed intakewas recorded.

HCA trisodium salt and HCAL aqueous solutions were adminis-tered to animals through stomach tubes. Feed intake and weightgain of 7 groups, namely, control group, three groups each ofHCA and HCAL fed with different molar concentrations (1.1, 3.7and 5.5 mmol/kg per day) were measured. Feed intake was moni-tored on a daily basis and calculated as total feed taken by each ratper week, whereas, weight gain was calculated as an averageweight gained by each rat at the end of every week. All the animalexperiments were conducted according to prescribed guidelineswith the approval of the concerned ethics committees.

3. Results and discussion

3.1. HCAL purity and stability

A simple and efficient alternative route has been described formaking high pure HCA derivatives avoiding ion-exchange chroma-tography. Crystalline HCAL can be made from either dried Garciniafruit rind or from commercially available HCA salts. Both sourcesoffer HCAL in pure form. Various metal salts with defined purityand composition have been made using this pure lactone. PureHCAL is crystalline and stable at room temperature under anhy-drous conditions for several months. When exposed to moisturein the atmosphere it was slowly converted into HCA and this con-version stopped after attaining the equilibrium of 60% of HCA to40% of HCAL which was monitored by HPLC. The rate of attainmentof equilibrium in water solution is temperature-dependent. Theequilibrium was reached within 4 h under acidic solutions and inaqueous solution maintained at about 80 �C but the conversionwas slow if the aqueous solution was kept at room temperature(Fig. 3). Acidification of HCA salts and HCAL with mineral acids alsoresulted in formation of an equilibrium mixture of HCA and HCAL.

3.2. Highly pure HCA salts

The pure HCAL obtained was reacted with different metalhydroxides and metal carbonates in stoichiometric quantities toget different salts of HCA with known purity and composition.

0

10

20

30

40

50

60

70

0h 1h 2h 3h 4h 5h 6h

Time

% o

f (-

) h

ydro

ctri

c ac

id

RT

50°C

80°C

Fig. 3. Conversion of HCAL into HCA in water at different temperatures.

Table 1HPLC and optical rotation data (in water) of HCAL at different temperatures.

Time in h 30 �C 50 �C 80 �C

%HCA %HCAL [a]D %HCA %HCAL [a]D %HCA %HCAL [a]D

0 1.4 98.6 +101.4 1.4 98.6 +101.4 1.4 98.6 +101.41 1.9 98.1 +101.3 5.4 94.6 +99.10 37.0 63.0 +73.72 2.6 97.4 +101.3 10.1 89.9 +95.0 48.4 51.6 +57.33 3.2 96.8 +101.0 13.6 86.4 +92.6 58.2 41.8 +51.24 3.7 96.3 +100.4 16.3 83.7 +90.0 60.6 39.4 +48.55 4.5 95.5 +100.2 19.8 80.2 +87.8 61.9 38.1 +47.26 5.1 94.9 +98.7 21.9 78.1 +83.9 62.2 37.8 +46.3

238 G. Venkateswara Rao et al. / Food Chemistry 120 (2010) 235–239

Different salts, such as trisodium, tripotassium, calcium–sodium,calcium–potassium and magnesium, were prepared and quantita-tively estimated for content of HCA using HPLC. Of all the salts pre-pared, the magnesium salt had the highest HCA content (84%) andthe tripotassium salt had the lowest (63%). Though all salts of HCAare moisture-sensitive, the tripotassium salt is highly hygroscopicin nature.

The specific rotations of mixtures of HCA and HCAL in differentratios were measured and there was linearity between % of HCAand specific optical rotation. As the concentration of HCA increasedwith decreasing amount of HCAL the specific optical rotation de-creased and these results match well with HPLC data (Table 1).Hence the optical rotation method would be helpful in estimationof the HCA content present in aqueous solutions.

3.3. Feed intake

The average feed intake per rat per week was measured over aneight week period and the results are incorporated in Table 2. De-crease in feed intake was observed in all rats treated with HCA andHCAL but it was more significant at higher molar concentrations.However, feed intake was lower in rats treated with HCAL than

Table 2Average feed intake in g per rat per week for eight weeks.

1 wk 2 wk 3 wk

Control group 122.5 111.2 112.5HCA (1.1 mmol) 108.2 95.6 96.2HCAL (1.1 mmol) 98.2 89.5 84.2HCA (3.7 mmol) 99.2 90.0 85.0HCAL (3.7 mmol) 86.2 85.7 78.2HCA (5.5 mmol) 95.0 83.2 79.5HCAL (5.5 mmol) 71.7 73.2 70.5

with HCA, in all dosage groups. Thus these results suggest thatHCAL has better appetite suppression activity than has HCA.

3.4. Increase in body weight

The average weight gained in the case of control rats (placebo)in the eight week period was found to be higher than in the case ofthose treated with HCA and HCAL (Table 3). As the molar concen-tration of HCA and HCAL increased, the reduction in weight gainwas significant. The HCAL-treated animals showed minimal weightgain in initial weeks and maximum weight loss at the end of thestudy when compared to HCA. Thus, the study reveals that HCALis acting better than HCA in controlling body weight in rats.

Commercial preparations of HCA are generally available as anaqueous concentrate or as salts, such as the calcium salt, cal-cium–sodium salt, and calcium–potassium salt. The active ingredi-ent HCA content in these preparations ranges between 50% and70% against 106% for the pure HCAL. Hence dosage, in terms ofquantity, would be significantly less in the case of HCAL. Moreover,HCAL, in an aqueous medium and under acidic conditions, attainsequilibrium with HCA. Hence it could be reasoned that HCAL whichgets converted into HCA may be responsible for the appetitesuppression.

4 wk 5 wk 6 wk 7 wk 8 wk

111.2 115.0 101.2 104.2 97.087.5 88.7 82.5 76.0 67.780.5 78.0 75.0 71.2 58.278.7 80.5 74.2 70.5 63.270.7 74.0 62.5 58.7 54.575.0 73.2 63.7 60.0 53.262.5 65.2 59.5 52.7 46.2

Table 3Weight gain in g per week for eight weeks (means ± SD).

1 wk 2 wk 3 wk 4 wk 5 wk 6 wk 7 wk 8 wk

Controla 24.5 ± 3.8 24.7 ± 6.3 12.7 ± 3.8 16.5 ± 3.4 19.0 ± 3.6 20.0 ± 5.5 10.0 ± 3.7 9.9 ± 3.3HCAb (1.1 mmol) 14.2 ± 3.0 23.7 ± 4.3 5.5 ± 2.4 11.2 ± 2.9 6.7 ± 0.5 9.0 ± 1.8 2.0 ± 0.8 �5.2 ± 2.54HCALc (1.1 mmol) 11.7 ± 4.9 19.2 ± 2.9 2.5 ± 1.0 7.2 ± 1.2 3.2 ± 1.2 0.2 ± 0.0 �7.7 ± 0.9 �7.2 ± 2.6HCAd (3.7 mmol) 10.6 ± 2.1 15.0 ± 4.1 4.0 ± 2.8 9.0 ± 4.8 3.5 ± 0.6 3.7 ± 0.9 �1.7 ± 0.5 �2.2 ± 0.9HCALe (3.7 mmol) 8.2 ± 3.3 13.2 ± 1.3 3.0 ± 1.8 5.7 ± 1.6 0.5 ± 0.0 �1.0 ± 0.7 �4.7 ± 1.4 �12.0 ± 4.3HCAf (5.5 mmol) 9.2 ± 1.5 14.5 ± 2.7 0.2 ± 0.1 8.2 ± 3.2 1.2 ± 0.6 0.2 ± 0.1 �6.2 ± 1.3 �10.5 ± 1.9HCALg (5.5 mmol) 6.0 ± 1.4 11.5 ± 3.4 0.7 ± 0.1 2.0 ± 0.8 �3.0 ± 0.8 �3.0 ± 0.8 �11.2 ± 2.9 �14.6 ± 5.9

a Average zero day body weight – 173.5 g.b Average zero day body weight – 157.75 g.c Average zero day body weight – 170.25 g.d Average zero day body weight – 178.5 g.e Average zero day body weight – 162.5 g.f Average zero day body weight – 144.0 g.g Average zero day body weight – 141.5 g.

G. Venkateswara Rao et al. / Food Chemistry 120 (2010) 235–239 239

3.5. Mechanism of action

HCA inhibits ATP–citrate lyase, a key enzyme responsible forpromoting fat synthesis. As a result of this inhibition, energy is di-verted for the production of glycogen in liver and muscles, insteadof fat synthesis. Since higher glycogen levels signal satiety to thebrain, HCA suppresses appetite. As a direct result of ATP–citratelyase inhibition, malonyl-CoA is not produced, which indirectlyhelps in burning the fat. The study of the mechanism of action ofHCAL would be difficult for the reason that it exists in equilibriumwith HCA under aqueous/acidic conditions. HCAL shows betteractivity in terms of appetite suppression and reduction in weightgain and this may be due to continuous conversion of HCAL intoHCA as it gets utilized by the body. HCAL may thus act as a pro-drug. Once HCAL gets converted into HCA it follows the samemechanism of action as HCA in reducing body weight gain andappetite suppression.

3.6. Safety

The age-old practice of consuming Garcinia fruit rind as a foodadditive by inhabitants of Malbar and the Konkan coast of the In-dian peninsula has established the safety of HCA. In free form,HCA exists as an equilibrium mixture of free acid and lactone,based on our experimental finding, and this scenario definitely ex-ists in naturally occurring fruits. Hence consumption of HCAL, orfruits as such, amounts to the same thing and perhaps HCAL wouldbe better as it can be taken at desired dosages.

4. Conclusion

A simple and efficient method has been developed for the prep-aration of HCAL and its salts. An HPLC method has been establishedto determine the purity and stability of these compounds in aque-ous solutions. Also, specific optical rotation has been employed forthe first time to estimate HCA and HCAL contents in solutions. Forthe first time we have established that free HCA exists in an equi-librium mixture of acid and its lactone in aqueous solution. Thepure HCAL shows better appetite suppression activity than do itssalts and hence can be used in food preparations and beveragesin place of HCA salts.

Acknowledgements

We thank Dr. Anil Kush, CEO, Vittal Mallya Scientific ResearchFoundation, for his encouragement and Dr. Latha Diwakara forhelpful discussions.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.foodchem.2009.10.014.

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