[77] Isolation and properties of hydroxycitric acid

[77]

ISOLATIONAND PROPERTIES OF HYDROXYCITRIC ACID

613

with 100 ml portions of hot ethyl acetate. The combined extracts are evaporated to dryness in vacuo at 40-50 ° to yield pure threo-isocitric lactone. The yield is 2.9 g (67.5~fl; B. P. 159.5-161°). The threo- and erythro-isocitric lactones can be identified by means of paper chromatography in the solvent system butanol-formic acidwater ( 4 : 1 : 2 ) . The spots are detected by spraying with 0.05% thymolblue in 94% ethanol. The R I values are 0.55 and 0.60 for the threo and erythro isomers, respectively. Addendum Isocitric acid was synthesized from sodium succinate and chloral, 4 and by the reduction of ethyl oxalosuccinate with sodium amalgam. 5 threo-n~(--)-Isocitric lactone was separated in considerable amounts from leaves of Bryophyllum calycinum by Pucher and Vickery, 6 and erythro-L,-(~-)isocitric lactone was separated from a culture of Penicillium purpulogenum var. rubrisclerotium Thom. No. 1148 by Sakaguchi. 7 R. Fittig and H. E. Miller, Ann. Chem. 25~ 43 (1889); H. A. Krebs and L. V. Eggleston, Biochem. J. 38, 426 (1944); G. W. Pucher and H. B. Vickery, J. Biol. Chem. 163, 169 (1946); H. P. Kato and S. R. Dickmann, Biochem. Prep. 3, 52 (1953). ~W. Wisleccnus and N. Nassauer, Ann. Chem. 285, 1 (1895); G. W. Pucher and H. B. Vickery, J. Biol. Chem. 163, 169 (1946). ~G. W. Pucher, J. Biol. Chem. 145, 511 (1942); G. W. Pucher, M. D. Abrahams, and H. B. Vickery, ibid. 172, 579 (1948). ~T. Beppu, S. Abe, and K. Sakaguchi, Bull. Agr. Chem. Soc. Japan 21, 263 (1957); K. Sakaguchi and T. Beppu, Arch. Biochem. Biophys. 83, 131 (1959).

[77]

Isolation and Properties of Hydroxycitric

Acid

B y Y. S. LEWIS Hydroxycitric acid (1,2-dihydroxypropane-l,2,3-tricarboxylic acid) has two asymmetric centers, hence two pairs of diastereoisomers or four different isomers (I, II, III, and IV) are possible. 1 Being a ~,-hydroxy acid, it cyclizes readily to the corresponding lactone. The relative rates of cyclization of the different isomers to the lactones are not known. 1The nomenclature for tile stereoisomers of hydroxycitric acid was kindly suggested by Dr. H. B. Vickery, Connecticut Agricultural Experimental Research Station, New Haven, Connecticut.

614

PREPARATION OF COMPOUNDS COOH

[77]

COOH

COOH

I

I H--C--OH

I

HO--C--H

I

H--C--OH

I

H O O C -- C-- O H

I

H O - - C -- C O O H

i

H O -- C -- C O O H

i

H-- C - - C O O H

I

H--C-- COOH

l

H - - C -- C O O H

l

H

l

H

H

(II)

(z)

COOH

(rII)

COOH

I

COOH

I

HO--C-- H

- -

l

[

l

C--H

i

C--H

f

,

HOOC - - C -- OH i H--C--COOH I

O HO--C--COOH I H--C--C--O i

O HOOC--C--OH I H--C--C-~-O J

(IV)

(v)

(Vl)

(i) (II) (III)

acid, erythro- Ds - h y d r o x y c i t r i c acid

Ds Dg - H y d r o x y c i t r i c

L s L g - H y d r o x y c i t r i c acid, erythro - Ls- h y d r o x y c i t r i c acid DsLg- H y d r o x y c i t r i c acid,

threo-Ds-hydroxycitric acid (IV)

L s D g - H y d r o x y c i t r i c acid,

threo-Ls-hydroxycitric acid (V)

L a c t o n e f r o m acid (II)

(VI)

L a c t o n e f r o m acid (IV)

Hydroxyeitrie acid was synthesized by Martius and Maue 2 from trans-aconitic acid by a modification of the method of Pawollek. s It was resolved chemically into its isomers. The natural occurrence of isomer IV of hydroxyeitric acid in the calyxes of Hibiscus sabdari]~a (Family Malvaceae) has been reported. '-8 More recently isomer (II) was isolated from the fruit rind of Garcinia cambogia (Family Gutti2C. Martius and R. Maue, Z. Physiol. Chem. 269, 33 (1941). 3A. Pawollek, Ann. Chem. 178, 155 (1875). ' C. Griebel, Z. Lebensm. Untersuch.-Forsch. 77, 560 (1939)-(Chem. Abslr. 33, 7491). C. Griebel, Z. Lebensm. Unters~ch.-Forsch. 83, 481 (1942)-(Chem. Abstr. 37, 4704). 6M. Bachstez, Ciencia Mex 9, 121 (1048); Chem. Abstr. 43, 7044 (1949).

[77]

ISOLATIONAND pROPERTIES OF tIYDROXYCITRIC ACID

615

ferae).7.8 The exact stereochemistry of either the synthetic or naturally occurring isomers is not known with certainty, and unequivocal methods are necessary to establish this aspect beyond doubt? The stereochemical structures (II and IV) suggested for the naturally occurring isomers are based on the existing chemical evidence. Hydroxycitric Acid from Garcinia The acid is present to the extent of 20-30% in the dried fruit rinds 1° of Garcinia cambogia, Garcinia atroviridis, and Garcinia indica, which are used for culinary purposes and are available commercially in India. 11 The acid can be isolated from these materials in the form of its lactonc by either of two methods. Method B is recommended, and Method A was employed during the original isolation, s

Method A Principle. Neutralization of concentrated extract of the acid in alcohol with K O H results in the separation of the potassium salt as a heavy oily liquid which is freed of impurities by repeated washing with alcohol. Solutions of the purified potassium salt are converted to the free acid on a cation exchange column. Evaporation of the aqueous solution of the acid yields the lactone (V). Procedure. Fruit rind of Garcinia cambogia (200 g) is autoclaved with 600 ml of water at 115 ° for 15 minutes. The cooled extract (25-30 °) is decanted through several folds of cheesecloth and filtered on a Bfichner funnel (Whatman No. 1 paper) ; the residue is washed with water. The dark brown filtrate (volume 600 ml) is concentrated to about 100 ml on a water bath and treated with 200 ml of ethanol with stirring. The resulting precipitate of pectinous material is removed either by centrifugation or filtration. The acidic filtrate is neutralized (pH paper) by cautious addition of 40% KOH, with careful stirring. The heavy oily ~Y. S. Lewis and S. Neelakantan, Current Sci. India 33, 82 (1964). Y. S. Lewis and S. Neelakantan, Phytochemi~try 4, 619 (1965). X-ray crystallographic studies with this goal are being pursued by collaborators of the late Dr. Patterson at the Institute for Cancer Research, Philadelphia, Pennsylvania. loThe commercially available fruit rinds of Garcirda cambogia are hard and dark brown in color. They contain considerable amounts of sodium chloride added during the curing procedure. Significant variations in the yield of hydroxycitric acid are not encountered with different batches of the material, ttydroxycitric acid was found to be the major organic acid present as determined by paper chromatographic examination. ~1Dried fruit rinds of Garcinia cambogia were purchased through the Pepper Research Station, Taliparamba, Kerala, India. For a description of Garcinia cambogia see "The Wealth of India (Raw Materials)," Vol. IV, p. 99. Council Sci. Ind. Res., New Delhi, India, 1956.

616

PREPARATION OF COMPOUNDS

[77]

liquid which is formed is allowed to settle for a few minutes and the supernatant is decanted and discarded. The oily liquid is washed with 60% ethanol (five portions of 100 ml). It is next washed with absolute alcohol (two portions of 100 ml), the suspension being left to stand for 60-90 minutes each time. A further portion of 100 ml of absolute ethanol is added and allowed to stand overnight. Ethanol is decanted, and the yellow (very hygroscopic) semisolid thus obtained is dried in vacuo at 80 ° to remove traces of ethanol and stored in a desiccator. The yield is about 40 g. A 10% solution of the salt (ca. 5 g) is passed through a column (20 X 3 cm) of cation exchange resin (e.g., Zeocarb-215). The column is washed with water until free of acid and the effluent is evaporated to dryness on a water bath. (A considerable amount of colored material is retained on the resin.) The residue is dried in a vacuum oven at 100 ° for 8-10 hours. A crude crystalline mass is obtained after the residue has stood in a desiccator for 7-8 days. Instead of being dried in vacuo, the residue (a thick sirup) can be seeded with a few crystals of the lactone to induce crystallization. The crude crystalline material (light brown in color) is further purified by extraction and recrystallization from ether. Method B Principle. The acid is extracted from the fruit rind with acetone. The acetone extract is concentrated, and the acid is taken up in water. The aqueous solution yields the crystalline lactone on evaporation. Procedure. Fruit rind of Garcinia cambogia (1 kg) is kept immersed in 1500 ml of acetone in a 4 liter flask and left overnight. It is reextracted with an equal volume of acetone in a similar manner. Acetone is removed from the combined extracts by distillation in vacuo. The viscous residue is stirred with a liter of water (45-50°), and the material is filtered through several folds of cheesecloth. The precipitated insoluble resinous material is thus removed. The reddish brown filtrate (80-90 ° ) is treated with activated charcoal (about 20 g) and concentrated to a thick sirup (light brown in color) on a water bath. It is seeded with a few crystals of the lactone and left overnight in a desiccator. A light brown, crystalline material is obtained. The yield is about 180 g. The material is vigorously extracted with 3 liters of ether (ten portions of 300 ml), and the combined extracts are dried over anhydrous sodium sulfate. A considerable proportion of the color is ether insoluble. The extract is decolorized with activated charcoal if necessary. Ether is then removed by distillation, and the sirupy mass thus obtained is heated as a thin layer on a water bath (10-15 minutes) to remove traces of ether. This results in a white solid. The yield is about 150 g.

[77]

ISOLATION AND PROPERTIES OF HYDROXYCITRIC ACID

617

Purification The lactone obtained by either of the above procedures is further purified by extraction with ether (1 g/20 ml) in several portions. The ether-soluble material is concentrated to one fourth its volume and an equal portion of dry chloroform is added with stirring. Upon standing, the lactone crystallizes as needles. The crystals are collected on a sintered glass funnel and are dried in vacuo. The pure material thus obtained has the properties described in the table. Hydroxycitric Acid (IV) from Hibiscus The acid is present in the calyxes of Hibiscus sabdarif]a. It is also found in the acidic leaves of Hibiscus cannabinus and Hibiscus ]urcatus. The best source for the isolation of the acid is the dried calyx of Hibiscus sabdari]Ja. 1~ A sample obtained from a crop grown in India contained 28% (dry weight basis) of the acid. The procedure described for its isolation is essentially similar to that of Griebel2 '~ Principle. The acid is extracted from the dried calyx powder with acetone. Water is not recommended as mucilaginous material is also extracted which makes further processing difficult. The acetone extract is concentrated, and the acid is taken up in water. It is precipitated as the lead salt and after removal of lead is isolated as the tactone from aqueous solution by concentration. Procedure. Powdered calyxes of Hibiscus sabdarif]a (100 g) were left immersed in 1 liter acetone at room temperature overnight. The suspension was filtered on a Biichner funnel (Whatman No. 1) and most of the acetone was removed by distillation in vacuo. The concentrate thus obtained was stirred with 500 ml of water and filtered on a Biichner funnel (Whatman No. 1 paper). The filtrate was treated with about 10 g of activated charcoal and filtered. The solution was neutralized (pH paper) with a saturated solution of neutral lead acetate. The insoluble lead salts were collected by eentrifugation and washed thoroughly with water to remove any excess lead acetate. A fine suspension of this material in water was treated with hydrogen sulfide. The lead sulfide was filtered off and the filtrate was evaporated to a sirupy consistency on a water bath. The semisolid material was kept in a desiccator for 2 or 3 days; during this time it solidified to a crystalline mass. The yield was about 20 g. Further purification was achieved by preparing a saturated solution of this material in ether; during this process impurities are eliminated. ~'~For a description of the plant, see J. M. Watt and M. G. Breyer Bradwyic, in "Medicinal and Poisonous Plants of Southern and Eastern Africa," p. 787. Livingstone, London, 1962.

618

[77]

PREPARATION OF COMPOUNDS

Addition of an equal volume of chloroform to the ether solution results in the crystallization of the product. Properties T h e pure lactones (V a n d VI) from acids I I a n d I V are hygroscopic, b u t are s t a b l e a t room t e m p e r a t u r e when k e p t in a desiccator. T h e i r general properties are listed in the table. Both the lactones form c r y s t a l PROPERTIES OF HYDROXYCITRIC ACID LACTONES

Property Melting point For free acidb After saturation with Boraxc Crystal shape Hygroscopicity Solubility

Susceptibility to purified threoD.-isocitrate: NADP oxidoreductase (decarboxylating) (EC 1.1.1.42), pig heart Paper chromatography R! valuesd BFW: Acid Lactone PAW: Acid Lactone Metavandate sprays Acid spot Lactone spot Quinine salt, m.p.

Hydroxycitric acid lactone (¥), Hibiscus lactone

Hydroxycitric acid lactone (VI), Garcinia lactone

183° +122 ° +31 ° +31 ° Needles High Very soluble in water and alcohol; slightly soluble in ether Negative

178° +100 ° --20 ° Needles Slight Very soluble in alcohol and water; fairly soluble in ether Negative

0.15 0.39 0.35 0.26

0.24 0.42 0.36 0.26

Yellow Yellow 227° decomp.

Reddish orange Yellow 215° decomp.

-

920

Two percent solution in water. The optical rotatory dispersion curves of both lactones indicate a positive Cotton effect with a maximum at 233 n ~ (W. Klyne, personal communication). b The lactones are warmed with sufficient equivalents of aqueous KOH for complete conversion to the tripotassium salt and passed through Zeocarb-215. Measurements of rotation and total acid are made on the column effluents. , Solutions of the acids, obtained as described in footnote b, are saturated by addition of solid Borax, and the rotations are measured. The values mentioned are not absolute but give an indication of the configuration. BFW: n-butanol-90% formic acid-water (4:1:5), Whatman No. 2 paper. PAW: n-propanol-ammonia-water (6:3:1), Whatman No. 2 paper. • H. A. Stafford, Am. J. Botany 46, 347 (1959).

[78]

HOMOCITRIC~HOMOACONITIC~ AND HOMOISOCITRIC ACIDS

619

line calcium salts (CaC~H~07"4 H20). The lactones give a positive test with hydroxylamine and can be assayed (0.1-1 /A~/) as their hydroxamates by a slight modification13 of the Lipmann-Tuttle procedure. 14 Hydroxyeitrie acid (IV) can be determined by a modification1~ of the metavanadate procedure. 15 Some preliminary work on the microbial metabolism of hydroxycitric acid (II) has recently been reported. 16 The structures predicted for the H i b i s c u s and Garcinia acids s have now been verified and found correct by X-ray diffraction studies carried out by Dr. Jenny Glusker, The Institute for Cancer Research, Philadelphia, Pennsylvania (private communication). Acknowledgment The author is indebted to Dr. P. R. Krishnaswamyand Dr. D. Rajagopala Rao for their assistance in the preparation of this article. ,3Unpublished procedure of D. R. Rao, 1965. "F. Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1945). "J. R. Matchett, R. R. Legavit, C. C. Nimmo, and G. K. Notter, Ind. Eng. Chem. 36, 851 (1944). "D. Rajagopal Rao and M. Ramakrishna, Biochem. Z. 344, 399 (1966).

[78] P r e p a r a t i o n of H o m o c i t r i c , H o m o a c o n i t i c , a n d Homoisocitric Acids

TuccI, Lovls N. CECI,and JNANENDRA K. BHATTACHARJEE

B y ANTHONYF.

Principle The methods for the preparation of homocitrie and homoaconitic acids are based on the work of Strassman and co-workers?,2 The synthesis of homoisocitric acid is based on the method of Yamashita2 Homoisocitric acid (I) is obtained by reduction of triethyl 2-oxaloglutarate, prepared by condensation of diethyl glutarate with diethyl oxalate in the presence of sodium ethoxide. The free acid is obtained from the triester of homoisocitrie acid by hydrolysis. Homoaconitic acids (II) are prepared by dehydrating triethyl homoisoeitrate with acetyl chloride, followed by hydrolysis to the free acids. The cis and t rans isomers of homoaconitic acid may be separated by anion-exchange chromatography; t r a n s - h o m o ' M. Maragoudakis and M. Strassman, 3. Biol. Chem. 241, 695 (1966). ' M. Stras~aan and L. N. Ceci, J. Biol. Chem. 241, 5401 (1966). s M. Yamashita, J. Org. Chem. 23, 835 (1958).