Studies on plant antibiotics - pterygospermin : The antibiotic principle of moringa pterygospermagaertn

Abstract

General Introduction 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, 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 micro organisms (which belong to lower plant life), have been amply demonstrated in recent years to offer a rich source of antibiotics; 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. Preliminary screening and examination of a crude extract of the roots of Wrightia tinctoria showed considerable promise. The present work on the chemical characterisation and investigations of the properties of the antibiotic designated “Pterygospermin” has been undertaken, and the results of these studies are now described in this thesis.

Botanical Notes Wrightia tinctoria, commonly known as the "dye yielding tree," has long been a favourite medicinal plant in indigenous medicine. Its botanical characteristics are given on page 24. The medicinal uses of various parts of the tree appear to involve the antibacterial substances, especially in the roots, which are used extensively to counteract septic and inflammatory conditions - a fact previously demonstrated in this laboratory through the preparation of a crude extract possessing pronounced antibacterial activity. A pure antibacterial agent has been isolated by the author, and knowledge as to its chemical nature has been obtained.

Section I To isolate and study the active principle, it was necessary to carry out systematic investigations into the best conditions for extraction and isolation. The effects of various factors such as seasonal variations, age of the tree, solvents, and temperature of extraction on the yield of the active material have been described. Chromatographic methods, solvent partitioning, removal of impurities by physical and chemical means, etc., have been studied in detail. It has been found that crude preparations of pterygospermin contain varying quantities of elemental sulphur, colouring matter, and phytosterols. After removal of these impurities, the purified pterygospermin (P.P.), on molecular distillation, gives rise to-by decomposition-benzyl isothiocyanate, which is also highly active. This aspect is discussed in Section III. The preparation of a low melting crystalline substance (m.p. 22-23°C) possessing the highest activity so far obtained has also been described. This substance is designated “Pterygospermin.”

Section III Physical and chemical properties-such as colour, odour, taste and appearance, coagulation point, density, refractive index, light absorption, solubility, thermal stability, stability in acidic and alkaline media, colour reactions-of the highly purified preparation of pterygospermin are given. Its antibacterial and antifungal activities are recorded (vide Tables XXII, XXIII, 69 and 71). Table XXIII contains activity data of crystalline pterygospermin.

Section IV The experiments conducted to elucidate the chemical nature of the active principle, pterygospermin, are fully described. Evidence indicates that the active principle is closely related to benzyl isothiocyanate. Points of similarity and differences between the two substances are discussed using chemical, physical, and biological evidence. Further, it has been found from analysis of different batches that purified pterygospermin generally contains varying amounts of the three derivative compounds:

Pterygospermin (Ia or Ib) Benzyl isothiocyanate oxide (IIa or IIb) Isothiocyanate (III)

Evidence for these is provided, including isolation procedures and reactions.

Section V A complete method of estimation of pterygospermin, benzyl isothiocyanate oxide, and benzyl isothiocyanate in various batches of purified material is given. It is based on independent estimation of:

Pterygospermin and benzyl isothiocyanate by iodine liberation from hydriodic acid Total isothiocyanate using a method analogous to Adams’ estimation of allyl isothiocyanate

Results for fourteen batches are summarised in Table XXIX (page 96). Great variations in composition are noted, making elucidation of the chemical nature of the active principle difficult.

Section VI This section summarises investigations designed to relate antibacterial activity to structural features of compounds related to pterygospermin and benzyl isothiocyanate. Many synthetic and naturally occurring compounds containing the structural sequence: -CH2-N=C=S-CH_2-N=C=S-CH2 -N=C=S or capable of releasing an isothiocyanate under biological conditions have been examined. The results suggest that the antibacterial activity of penicillin and gliotoxin may also be related to the presence of this structural arrangement. Its significance in explaining varying activities across penicillin types is discussed (pages 103-105).

Section VII The action of pterygospermin and benzyl isothiocyanate on germination of seeds (both monocots and dicots) has been studied. A strong inhibitory effect at high concentrations (100 ppm) is observed in barley, ragi, and horsegram; moderate in paddy and blackgram. No selective action between monocots and dicots is evident. Possible applications in controlling seed pests or in food preservation (due to low toxicity and pleasant aroma) are suggested. Glutamic acid counteracts the inhibitory effect, while thiamine does not.

Section VIII The influence of various amino acids, vitamins, and growth factors on the action of the antibiotic has been investigated (vide Tables XLIX and LII, pp. 133-134). Thiamine, aspartic acid, and pyridoxine reinforce the action of pterygospermin. The antibiotic is more stable in phosphate buffer (pH 7.0) than in distilled water. Cobalt has no effect.

Section IX The assimilation of glutamic acid by Micrococcus pyogenes var. aureus is affected by the antibiotic. Cells previously exposed to the antibiotic fail to utilise glutamic acid. This has been interpreted in terms of reversible combination of the antibiotic with the prosthetic group (pyridoxal phosphate) of the transaminase system. This mechanism helps to explain the reinforcing effect of pyridoxine and the antagonism of glutamic acid.

Section X The acute and chronic toxicity of purified pterygospermin, benzyl isothiocyanate (B.I.T.C.), and the bisulphite addition compound of benzyl isothiocyanate (B.A.C.) in mice and rats, by subcutaneous and oral administration, has been studied.

Pterygospermin is far less toxic than benzyl isothiocyanate. B.A.C. is well tolerated even at high doses. Pterygospermin and B.A.C. lower blood pressure and depress respiration and cardiac function, whereas B.I.T.C. increases blood pressure at high doses.

Protection experiments against experimental Staphylococcus and plague infections in mice show that P.P. and B.A.C. are protective in staphylococcal infections but ineffective in plague.