Chemical and biological investigation on antibiotic principles of Garcinia Morella

Abstract

The thesis comprises two parts. Part I describes in four chapters the isolation and chemistry of some of the constituents, and Part II describes their biological properties. Part I. Chapter I: Isolation and Properties of Guttiferin Introduction: The earlier work on the isolation, chemistry and biological properties of the compounds from the seed coat of Garcinia morella and commercial gamboge (resinous exudation) has been critically reviewed. The substances characterized may be grouped according to their acidic or neutral character into guttiferin or morellin series. Attention has been focussed on the possibility of some of the compounds earlier characterized-particularly those displaying little or no antimicrobial activity-being artefacts formed during their isolation. The structures of different compounds of the two groups, viz., isomorellin, desoxymorellin, dihydroisomorellin, ethoxydihydroisomorellin and guttiferin ( gambogic acid) have been postulated mainly based on the elucidation of the structure of p bromobenzene sulphonyl derivative of morellin, which has yet to be established unequivocally as a true sulphonyl derivative of morellin. In view of these earlier studies, the need arose for a re examination of the earlier isolation procedures vis à vis retention of the various biological responses by the seed coat and gamboge preparations, which include cathartic activity. Accordingly, the object of the present investigations on the chemical and biological properties of the constituents has been defined. Isolation and properties of guttiferin: Direct extraction of the seed coat with ether instead of hot ethanol or acetone, followed by separation of the acidic guttiferins by extraction with sodium carbonate, crystallisation from aqueous pyridine and decomposition of the pyridine salt m.p. 115–117°C by hydrochloric acid gave an guttiferin m.p. 113–115°C different from the one previously described by Nageswara Rao and Narasimha Rao in respect of ultraviolet absorption characteristics (280, 290 and 358 mµ; for the earlier reported one 278 and 360 mµ). It also differed in being a potent cathartic. Since it is readily isomerised to the known compound by treatment with ethanol, the two isomers are now designated and guttiferins. The two guttiferins, however, give rise to the same methyl guttiferinate and guttiferic acid. Further, their hexahydro derivatives seem identical. The guttiferins display qualitatively and quantitatively similar antibacterial and antiprotozoal activity, which are discussed in Part II.

Part I. Chapter II: Structure of and Guttiferins, Guttiferic Acid and Compound C H O The chemical and spectroscopic evidence, which includes UV, IR, NMR and mass spectral characteristics, leading to the postulation of the structure of guttiferin, have been discussed in light of earlier formulations of morellin and guttiferin ( gambogic acid). The structure of guttiferin follows that of guttiferin, which is the stable trans isomer being readily formed from it in contact with ethanol, and is thus assigned cis configuration of the unsaturated carboxylic group and the proton in the side chain. Further, the structure of guttiferic acid (C H O ), produced from both guttiferins on treatment with alkali and presently obtained in a crystalline state, has been discussed. It is shown to be analogous to hydrogambogic acid (garcinolic acid, C H O ) derived from guttiferin by fission of the cyclic ketone. The substance previously termed as an acidic phenol by Krishnamurthy and Narasimha Rao (obtained by alkaline degradation of octahydromorellin) has been elucidated. This substance is also formed from hexahydro guttiferin under similar conditions, but not from octahydro guttiferin. NMR data and its properties suggest that it may be a tautomeric mixture (A or B). Its monomethyl ether corresponds accordingly. The course of degradation of guttiferin derivatives leading to formation of phloroglucinol, methylheptenol, homophthalic acid, isovaleric and isocaproic acids and acetone, shared by guttiferin, has been suggested.

Part I. Chapter III: Guttiferin ( Gambogic Acid) – the Cathartic Principle of Gamboge In an attempt to detect and isolate an isomer of guttiferin from commercial gamboge, it is found that guttiferin forms highly characteristic crystalline complexes with one molecule of DMF, DMSO, and THF. While the known pyridine complex is a true salt, the above complexes differ in characteristics. The mechanism of complexation appears to involve hydrogen bonding with atoms having lone pairs. Evidence shows oxygen participates in complex formation, not nitrogen or sulphur. The complexes dissociate in solution and displace each other. Biological evidence also suggests dissociation during activity. Other guttiferins ( , , , ) do not form these complexes.

Part I. Chapter IV: Guttiferin – the Antiprotozoal Principle of Gamboge Gupta and Narasimha Rao found that the residue after crystallisation of guttiferin–pyridine salt contained potent antiprotozoal substances. Three fractions- , and guttiferins-were eluted. guttiferin (C H O ) is essentially homogeneous. It analyses for tetrahydro guttiferin. UV absorption similarities suggest a shared chromophore with guttiferin. More detailed analysis is still needed. Its derivatives (methyl ester, guttiferic acid) crystallise readily. Guttiferin shows high antiprotozoal activity-2.5× that of guttiferin.

Part II: Antimicrobial and Cathartic Activity of and Guttiferins and Derivatives The methods for antibacterial, antifungal and antiprotozoal assays, toxicity, cathartic activity and anti staphylococcal activity in mice have been described. The data indicate:

and guttiferins behave similarly to morellin analogues. Strong activity against Gram positive bacteria. Little or no action on Gram negative bacteria or fungi. Bacillus subtilis is highly sensitive (MIC 0.2–0.4 g/mL). Their activity is not significantly affected by glucose. Unlike morellin, and guttiferins are less affected by serum. Methionine and ferrous ions counteract activity; cysteine enhances it.

Structure–activity: A key finding is that cis trans isomerisation does not affect antibacterial activity (though it changes cathartic action). This differs from morellin. Antiprotozoal activity: Guttiferin has >2× the activity of guttiferin on Paramecium caudatum. Cathartic action: Guttiferin is established as the cathartic principle of gamboge. Guttiferins lack cathartic action. Toxicity: LD values in mice indicate high sensitivity via intraperitoneal administration. Both and guttiferins protect mice in experimental staphylococcal infections.