Studies on the antibiotic principles of plant resins from garcinia morella and eucalyptus citriodora

Abstract

The thesis consists of five chapters.

Chapter I A short résumé of the salient developments in the field of antibiotics since the discovery of Penicillin is given. The definition of the term “antibiotic” has been fully discussed. Further, the production of antibiotics by bacteria, actinomycetes, moulds and fungi, algae, lichens, yeasts, and higher plants has been described. A broad chemical classification of the antibiotics is also included. In this connection, it has been mentioned that higher plants-like the micro organisms (which belong to lower plant life)-have been amply demonstrated in recent years to offer a rich source of antibiotics, and in many cases substances of very similar chemical nature (vide pages 10-11) are produced by both plants and micro organisms. The present work forms part of a programme of research on “Plant Antibiotics” that is in progress in this laboratory. These investigations were undertaken with a view not only to evolve therapeutically useful substances from indigenous plant sources but also to substantiate scientifically the claims of practitioners of the indigenous system of medicine in so far as they relate to the treatment of microbial infections. In this connection, a study of the extensive indigenous practice of using fresh plant saps, exudations and resinous materials (vide Chapter II) in the prevention and treatment of bacterial infections has been undertaken. As a preliminary step, a few of these materials have been screened. Two of them, namely gamboge from Garcinia morella and kino from Eucalyptus citriodora, have been investigated in detail, and the results are described in this thesis.

Chapter II By way of introduction, a short résumé is given as to the classification of naturally occurring resins, their botanical constitution, technological applications and the medicinal virtues commonly attributed to them. The chemistry of resins and their constituents has been discussed as a preliminary appraisal of their behaviour vis à vis their antibacterial activity (cf. Chapter III). The general characteristics of eight representatives of this class, commonly available in this country, are given. The analytical methods employed for this purpose have also been discussed.

Chapter III After considering various methods and procedures available for qualitative and quantitative assaying of substances for their antibacterial activity, it has been suggested that the usual serial dilution method, with a few modifications, is suitable for assessing the antibacterial activity of resins and their constituents. The details of this procedure are fully discussed. The results of assay of the following resins, plant exudations, and other plant parts, as well as well known constituents of resins and related substances, are given (vide Tables VI to XLIII, pages 63-82):

Gamboge, Shorea robusta, Vateria indica, Pinus excelsa, Garcinia indica, Canarium strictum, lac, Ferula asafoetida, kino from Eucalyptus citriodora, wax from the leaves of Santalum album, and juice of Carica papaya Abietic acid, dehydro abietic acid, maleic anhydride adduct of abietic acid, dibromomaleic anhydride adduct of abietic acid, triamide of maleic anhydride adduct of abietic acid, hydroxy abietic acid, d pimaric acid, maleic anhydride adduct of abietene Unsaponifiable matter of Pinus excelsa (vide page 46) Ether soluble and ether insoluble triterpene acids from Shorea robusta and resin oil of Shorea robusta Resin oil and polymerised resin oil of Vateria indica and phenols of Vateria indica Extracts from fruit, pulp, bark and leaves of (a) Garcinia cambogia, (b) G. indica, and (c) G. morella

Chapter IV In view of the high activity of gamboge-the resinous exudation from Garcinia morella-it has been examined in detail. This substance is of great interest in that our colleagues in this laboratory have established the antibacterial activity, therapeutic usefulness and chemical nature of morellin, a crystalline antibiotic present in the pericarp of the seeds of this tree. Investigations on morellin by colleagues, as well as work on resinous exudations from related species (especially G. hanburii, which yields the gamboge formerly included in B.P.), have been discussed. It has been pointed out that as the constituents of gamboge are highly sensitive to various reagents, especially alkalis, previous reports on separation of various amorphous constituents of gamboge from G. hanburii leave much to be desired. Accordingly, suitable methods had to be devised for their isolation and investigation. By a combination of chromatographic procedures, solvent partitioning, absorption characteristics, optical rotation and antibacterial activity as criteria for separation, a procedure has been described whereby highly purified-though amorphous-fractions possessing exceptionally high activity of the same order as morellin have been isolated. A preliminary report on some of their pharmacological properties is included. Chemically, it has been shown that Fraction VI (vide Table XIV, page 118) and “ garcinolic acid” (obtained by the lead acetate purification method without previous alkali treatment) contain a major constituent closely related to morellin, as they yield on alkaline degradation: (i) phloroglucinol (ii) homophthalic acid (iii) methyl heptenol Since structural alteration may occur during chromatography (as in the case of morellin), Fraction VI (but not necessarily garcinolic acid) may be an artefact. To distinguish clearly, the name Guttiferin is suggested for the active substance present in gamboge. In view of the ready solubility of guttiferin fractions (unlike morellin) in aqueous sodium carbonate (0.1%) but not in sodium bicarbonate, and based on their physico chemical properties, a general structural formula (I) has been proposed to explain their behaviour. However, this is not to be construed as the definitive structure, which must await isolation in crystalline form. Using a similar procedure, it has been shown that guttiferin is probably distributed throughout the plant, since extracts from bark, fruit rind, seed coat and leaves give fractions very similar to Fraction VI. The results are given separately in Sections B to E of this chapter.

Chapter V This chapter describes the results of further examination of the kino from Eucalyptus citriodora, which showed activity against both Gram positive and Gram negative organisms. It is noted that this kino is an interesting example of a plant product which, though showing a low titre in screening tests, actually contains a highly potent substance in low concentration-an important observation, since usual pharmacological methods might easily overlook such substances. After reviewing the general nature and production of kinos by various plants and prior work on their classification and chemistry, a method for concentrating the substance active against M. pyogenes var. aureus has been worked out. During this procedure, three crystalline substances have been isolated:

7 methyl kaempferol (definitely identified) A higher saturated hydrocarbon A third compound tentatively identified as 7 methyl aromadendrin based on analysis and physico chemical data

The active substance, designated “Citriodorol”, could not be obtained in crystalline form despite its high antibacterial activity. Based on its properties and absorption spectrum, it is likely related to pinoresinol, sesamine and nordihydroguaiaretic acid. Additionally, this is the first report of the occurrence of 7 methyl kaempferol and 7 methyl aromadendrin in any Eucalyptus kino and the first identification of a sugar component (glucose) in any kino-findings of considerable significance for biogenesis, kino classification, and evolutionary classification of Eucalyptus species.