C₁–C₆ Monocarboxylic acids

A number of these acids together with hydroxy- and keto-derivatives are intermediates in the early stages of the biosynthesis of fats, isoprenoid compounds and various amino acids (q.v.). In the free state they are not found abundantly in nature but occur scattered throughout the plant kingdom in the esterified form as a feature of some volatile oils, resins, fats, coumarin derivatives and alkaloids.

Some common acids are illustrated in Table 19.1.

Table 19.1 Examples of C₁–C₆ monocarboxylic acids.

Name	Structure	Comments
Formic acid		Name derives from its first isolation from the ant, Formica rufa. A decomposition product of many vegetable materials. Occurs free in the hairs of the stinging nettle; combined in the gitaloxigenin series of cardioactive glycosides. N-formyl-L-methionine is involved in the initiation of protein synthesis on ribosomes
Acetic acid		An essential primary metabolite, particularly as acetyl-CoA. Common in the esterified form
Propionic acid		Produced in the fatty acid oxidative cycle when an acyl-CoA with an odd number of carbon atoms is involved. Esterified as a tropane alkaloid
n-Butyric acid iso-Butyric acid		Occurs in traces in many fats Occurs free in carob beans (Ceratonia siliqua) and as its ethyl ester in croton oil. Component of resins of the Convolvulaceae and minor tropane alkaloids. Intermediate in the metabolism of valine
n-Valeric acid		Not common; component of Convolvulaceous resins
iso-Valeric acid		Free and esterified in Valeriana spp. Combined in some tropane alkaloids (e.g. valeroidine) and in the pyranocoumarin, dihydrosamidin. Intermediate in the metabolism of leucine
2-Methylbutyric acid		Component of some tropane and Veratrum alkaloids, Convolvulaceous glycosides and the pyranocoumarin visnadin
Caproic acid		Occurs in traces in many fats
Crotonic acid (trans– butenoic acid)		Constituent of croton oil
Tiglic acid		Occurs in croton oil (glycoside) from Croton tiglium. The acid of many minor tropane alkaloids, e.g. tigloidine. Component of Convolvulaceous resins and Symphytum alkaloids. Biosynthetically derived from isoleucine
Angelic acid		Occurs in the rhizome of Angelica. Esterifying acid of the Schizanthus alkaloid schizanthine X and of some volatile oils, e.g. chamomile oils. Component of the Cevadilla seed alkaloid cevadine and Symphytum alkaloids
Senecioic acid		First isolated from a species of Senecio (Compositae). Occurs as the esterifying acid of some alkaloids of Dioscorea and Schizanthus. Component of the pyranocoumarin samidin

Fatty acids

These acids are important as components of plant oils (acyl lipids) in which they occur as esters with the trihydric alcohol glycerol. They are also components of the resins of the Convolvulaceae and of waxes in which they are esterified with long-chain alcohols. Most are C₁₀ to C₂₀straight-chain monocarboxylic acids with an even number of carbon atoms. Over 200 have been isolated from natural sources but relatively few are ubiquitous in their occurrence. They may be saturated (e.g. palmitic and stearic acids) or unsaturated (e.g. oleic acid). The double bonds, with a few minor exceptions such as the seed oil of pomegranate, are cis.

Less commonly they are cyclic compounds such as hydnocarpic acid and the prostaglandins. The latter are a group of physiologically active essential fatty acids found in most body tissues and are involved in the platelet-aggregation and inflammatory processes. They promote smooth muscle contraction making them of clinical use as effective abortifacients and for inducing labour. All the active natural prostaglandins are derivatives of prostanoic acid (see Table 19.4). A rich source of prostaglandin A₂ (PGA₂) is the soft coral Plexaura homomalla. Although recognized in the 1930s, and their structures determined in 1962, it was not until 1988 that prostaglandins were unequivocally established as components of some higher plants (cambial zones and buds of Larix and Populus spp.)

Table 19.4 Cyclic unsaturated acids.

Common name	Structural formula
Hydnocarpic
Chaulmoogric
Gorlic
Prostanoic
PGA2

The characteristic acid of castor oil, ricinoleic acid (hydroxyoleic acid) has both a hydroxyl group and an unsaturated double bond. A range of acetylenic fatty acids occurs throughout the plant kingdom and some of them possess antifungal and antibacterial properties. The biogenetic relationship between these, the olefinic fatty acids and the saturated fatty acids is outlined later in this chapter.

Examples of fatty acids are listed in Tables 19.2–19.4. It will be noted that some have more than one unsaturated bond, the bonds being interspersed by methylene groups. These polyunsaturated acids have received much attention in recent years both regarding their role in dietary fats and as medicinals. All the common acids have trivial names but in order to indicate more precisely their structures without recourse to the full systematic chemical name each can be represented by a symbol. Thus α-linolenic, systematic name all-cis-Δ^9,12,15-octadecatrienoic acid, has 18 carbons and three double bonds which can be represented by 18:3. The position of the double bonds is then indicated by the n-x convention where n = number of carbon atoms in the molecule and x is the number of inclusive carbon atoms from the methyl (ω) end to the first carbon of the first double bond, in this case 3, so that the symbol for α-linolenic acid is 18:3(n-3). The positions of the two remaining double bonds are deduced by the fact that they will follow on from each other being separated only by one methylene (-CH₂-) group. In this area students may find the literature situation somewhat confusing because in some texts the acids may be symbolized on the basis of conventional chemical systematic numbering—for fatty acids the carboxyl carbon being C1. For α-linolenic acid this is represented as 18:3(9c.12c.15c), c indicating a cis-bond. The advantage of the first system is that it indicates any bioequivalence of the double bonds in acids of different chain-length, bearing in mind that chain elongation in vivo proceeds at the carboxyl end of the molecule by the addition of 2C units. Thus it can be seen from Table 19.3 that γ-linolenic acid and arachidonic acid both fall into the biochemical ω-6 family of unsaturated fatty acids and their respective symbols 18:3(n-6) and 20:4(n-6) reflect this whereas symbols based on chemical nomenclature for these acids viz 18:3(6c,9c,12c) and 20:4(5c,8c,11c,14c) do not. A comparison of symbols for some common unsaturated acids is shown in Table 19.5.

Table 19.2 Straight-chain saturated acids.

Table 19.3 Straight-chain unsaturated acids.

Table 19.5 Comparison of symbols ascribed to unsaturated fatty acids.

Under certain conditions, which are specified in pharmacopoeias, iodine or its equivalent is taken up at these double bonds and the so-called iodine value is thus a measure of unsaturatedness. The iodine value is the number of parts of iodine absorbed by 100 parts by weight of the substance. Near-infrared spectroscopy can also be used todetermine this value as it is directly related to the HC=CH stretch bands at 2130 nm in the spectrum. Iodine values are useful constants for acids, fixed oils, fats and waxes, and help to indicate the composition of complex mixtures as well as pure substances.

Biosynthesis of unsaturated fatty acids

Before the elucidation of the overall chemistry of formation of polyunsaturated fatty acids such as linoleic in the early 1960s by Bloch, knowledge concerning the biosynthesis of these compounds lagged behind that of the saturated acids. Recent progress has been much more rapid and, in general, it now appears that in aerobic organisms, monoenoic acids with the double bond in the 9,10-position arise by direct dehydrogenation of saturated acids. In higher plants, for this reaction, coenzyme A may be replaced by the acyl carrier protein (ACP), and Bloch has demonstrated that stearoyl-S-ACP is an effective enzyme substrate of the desaturase system of isolated plant leaf chloroplasts. The reduced forms of nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen are cofactors.

The position of the introduced double bond in respect to the carboxyl group is governed by the enzyme; hence, chain length of the substrate acid is most important. The hydrogen elimination is specifically cis but a few unusual fatty acids such as that in the seed oil of Punica granatum with the structure 18:3 (9c,11t,13c) have trans bonds. As illustrated in Fig. 19.1, further double bonds may be similarly introduced to give linoleic and linolenic acid.

Fig. 19.1 Sequence of formation of olefinic fatty acids in plants.

Unsaturated fatty acids can also be formed in plants by elongation of a medium-chain-length unsaturated acid. This appears to occur by the formation of an intermediate, β,γ-unsaturated acid rather than the α,β-unsaturated acid normally produced in saturated fatty acid biosynthesis; the β,γ-bond is not reduced and more C₂ units are added in the usual way (Fig. 19.2).

Fig. 19.2 Alternative pathways for synthesis of unsaturated fatty acids.

Sterculic acid, a component of seed oils of the Malvaceae and Sterculiaceae, is a cyclopropene and is also derived from oleic acid, with methionine supplying the extra carbon atom to give, first, the cyclopropane. Ricinoleic acid is a hydroxy fatty acid found in castor oil seeds and is again biosynthesized from oleic acid (Fig. 19.3).

Fig. 19.3 Oleic acid as the precursor of ricinoleic and sterculic acids.

Some of the natural acetylenes and acetylenic fatty acids have obvious structural similarities to the more common fatty acids. The hypothesis that triple bonds are formed from double bonds by a mechanism analogous to that for the formation of double bonds and involving structurally and stereochemically specific enzymes has now received experimental support. By this means (Fig. 19.4) the range of acetylenes found in Basidiomycetes and in the Compositae, Araliaceae and Umbelliferae can be derived from linoleic acid via its acetylenic 12,13-dehydroderivative, crepenynic acid, an acid first isolated from seeds oils of Crepis spp.

Fig. 19.4 Formation of acetylenic fatty acids.

Aromatic acids

Two common aromatic acids are benzoic acid and cinnamic acid (unsaturated side-chain), which are widely distributed in nature and often occur free and combined in considerable amounts in drugs such as balsams. Truxillic acid, a polymer of cinnamic acid, occurs in coca leaves. Other related acids of fairly common occurrence are those having phenolic or other groupings in addition to a carboxyl group; such are: salicyclic acid (o-hydroxybenzoic acid), protocatechuic acid (3,4-dihydroxybenzoic acid), veratric acid (3,4-dimethoxybenzoic acid), gallic acid (3,4,5-trihydroxybenzoic acid) and 3,4,5-trimethoxybenzoic acid. Similarly, derived from cinnamic acid, one finds p-coumaric acid (p-hydroxycinnamic acid), ferulic acid (hydroxymethoxycinnamic acid), caffeic acid (hydroxycinnamic acid) and 3,4,5-trimethoxycinnamic acid. Unbelliferone, which occurs in combination in asafoetida, is the lactone of dihydroxycinnamic acid.

Acids having an alcohol group are quinic acid (tetrahydroxyhexahydrobenzoic acid), which occurs in cinchona bark and in some gymnosperms; mandelic acid, C₆H₅CHOHCOOH, which occurs in combination in cyanogenetic glycosides such as those of bitter almonds and other species of Prunus; and shikimic acid, an important intermediate metabolite (see Fig. 18.8). Shikimic acid has itself acquired recent pharmaceutical importance as the starting material for the semi-synthesis of the antiviral drug oseltamivir (Tamiflu®) for use against bird flu infections in humans. Its principal source has been star-anise fruits (q.v.), leading to a supply shortage of the plant material. Other natural sources rich in this acid and of potential future use are needles of the Pinaceae (S. Marshall, Pharm. J., 2007, 279, 719) and the fruits (gumballs) of the American sweet gum tree Liquidamber styraciflua (q.v.). The acid is also produced commercially by the fermentation of genetically modified Escherichia coli. Tropic acid and phenyllactic acid are two aromatic hydroxy acids that occur as esters in tropane alkaloids (q.v.). For examples of the above see Fig. 19.5.

Fig. 19.5 Aromatic and other cyclic acids.

Chlorogenic or caffeotannic acid is a condensation product of caffeic acid and quinic acid. It occurs in maté, coffee, elder flowers, lime flowers, hops and nux vomica and is converted into a green compound, which serves for its detection, when an aqueous extract is treated with ammonia and exposed to air. See also ‘Pseudotannins’ (Chapter 21).

The biogenesis of the aromatic ring has been discussed in Chapter 18.

Monohydric aliphatic alcohols

Lower members of the series are found principally combined as esters e.g. methyl salicylate in oil of wintergreen and methyl and ethyl esters responsible for some fruit aromas. Esterified long-chain alcohols are constituents of some pharmaceutically important animal waxes and include cetyl alcohol (C₁₆H₃₃OH), ceryl alcohol (C₂₆H₅₃OH) and myricyl alcohol (C₃₀H₆₁OH). Such alcohols also participate in the formation of esters which are constituents of leaf cuticular waxes; an example is Carnauba Wax BP which contains myricyl cerotate.

Monohydric terpene alcohols

These are alcohols associated with that large group of compounds which arise from mevalonic acid and have isoprene as a fundamental structural unit. Pharmacognostically they are particularly evident as constituents of volatile oils namely: (1) non-cyclic terpene alcohols occur in many volatile oils—for example, geraniol in otto of rose, its isomer nerol in oils of orange and bergamot and linalol both free and combined as linalyl acetate in oils of lavender and rosemary; (2) monocyclic terpene alcohols are represented by terpineol and its acetate in oil of neroli and menthol and its acetate in oil of peppermint; (3) dicyclic terpene alcohols are particularly abundant in the Coniferae (e.g. sabinol and its acetate in Juniperus sabina). In the dicotyledons oil of rosemary contains borneol and its esters.

Monohydric aromatic alcohols

Benzyl alcohol, C₆H₅CH₂OH, and cinnamyl alcohol, C₆H₅CH= CHCH₂OH, occur both free and as esters of benzoic and cinnamic acids in balsams such as Tolu and Peru. The latter balsam is sometimes adulterated with cheap synthetic benzyl benzoate.

Included in this class is coniferyl alcohol, which forms an important component of the lignin molecule. Lignins are extremely complex phenylpropane polymers; they form an important strengthening material of plant cell walls and vary in composition according to their source, see Chapter 21, section on ‘Lignans and Lignin’.

Dihydric alcohols

Dihydric alcohols or glycols are compounds containing two hydroxyl groups; they are found naturally in many structural classes of compounds. The bicyclic amino alcohol 3,6-dihydroxytropane occurs free and as esters in a number of species of the Solanaceae and Erythroxylaceae, the dihydric alcohol panaxadiol is a component of some ginseng steroids, and oenanthotoxin, the poisonous constituent of the hemlock water dropworts (Oenanthe spp.), is a polyene diol.

Trihydric alcohols

As with the glycols these compounds occur in a range of structural types. An important example is glycerol (propan-1,2,3-triol), an essential component of fixed oils and fats which are discussed in more detail below.

Polyhydric aliphatic alcohols

The following are alcohols with either four or six hydroxyl groups. The meso form of erythritol, CH₂OHCHOHCHOHCH₂OH, is found in seaweeds and certain lichens both free and combined with lecanoric acid. The hexahydric sugar alcohols (e.g. sorbitol, mannitol and dulcitol) are formed in nature by the reduction of either an aldehyde group of an aldose or the keto groups of a ketose. Sorbitol is abundant in many rosaceous fruits, mannitol in manna and dulcitol in species of Euonymus.

Fats and fixed oils

As agricultural crops, seeds used for the extraction of fixed oils rate in importance second only to cereals. Over the last 60 years the production of oils for the food industry has increased enormously, whereas consumption by industrial and other users has remained relatively static but, in the pharmaceutical industry at least, not without interesting developments. Fixed oils are also obtained from fruit pericarps and in some instances such as the palm, Elaeis guineensis (Palmae), two oils differing in properties and chemical composition are obtained—the pleasantly flavoured palm kernel oil from the endosperm and palm oil from the orange-yellow fleshy pericarp. Oil seed crops are particularly advantageous commercially as following the expression of the oil a valuable high protein cattle feed remains. Also, such crops have benefited from plant breeding both regarding the yield and nature of the oil produced, and the morphology of the plant itself (see Chapter 14).

A naturally occurring mixture of lipids such as olive oil or oil of theobroma may be either liquid or solid and the terms ‘oil’ and ‘fat’ have, therefore, no very precise significance. Coconut oil and chaulmoogra oil, for example, leave the tropics as an oil and arrive in Western Europe as a solid. Even an oil such as olive oil will largely solidify in cold weather. In general, acylglycerols involving saturated fatty acids are solid and those of unsaturated acids are liquids. When both types are present, as in crude cod-liver oil, cooling results in the deposition of saturated acylglycerols such as stearin. In most medicinal cod-liver oils these solid materials are removed by freezing and filtration. Acylglycerols are represented by the general formula given below and can be hydrolysed by heating with caustic alkali to form soaps and glycerin.

If the fatty acids represented by R¹, R² and R³ are the same, the triacylglycerol is known as a simple triacylglycerol—for example, tripalmitin on hydrolysis yields three molecules of palmatic acid. In nature, however, R¹, R² and R³ are usually different and the ester is known as a mixed triacylglycerol. On hydrolysis they frequently yieldboth saturated and unsaturated acids (Fig. 19.6); there is a strong tendency for unsaturated acids, particularly the C₁₈ olefinic acids, to be linked to the secondary hydroxyl.

Fig. 19.6 Hydrolysis of a mixed triacylglycerol.

Biogenesis

Acylglycerols are formed from the fatty acyl-CoA or, more probably, the fatty acyl carrier protein (ACP) and L-α-glycerophosphate, as indicated in Fig. 19.7.

Fig. 19.7 Formation of acylglycerols; R¹, R² and R³ may or may not be the same acyl groups.

Quantitative tests

A number of quantitative tests are commonly used to evaluate fixed oils and fats. Acid value refers to the number of mg of potassium hydroxide required to neutralize the free acids in 1 g of the oil; high acid values arise in rancidified oils. Particularly low values are officially specified for those oils to be used in parenteral dosage forms; for refined wheat-germ oil, for example, the value is 0.3, whereas for the refined oil for general use the value is 0.9 and for the unrefined oil 20.0. Similar figures apply to other fixed oils so used. Saponification value: the hydrolysis reaction of lipids (above) can be used to determine the saponification value of the oil and is expressed as the number of mg of potassium hydroxide required to neutralize the free acids in, and to hydrolyse the esters in, 1 g of the substance. Ester value is the difference between the saponification and acid values. Iodine value (see ‘Fatty acids’) gives a measure of the unsaturation of the oil. Oils which partially resinify on exposure to air are known as semidrying or drying oils. Such oils (e.g. linseed oil) have high iodine values. In some cases, particularly for animal fats such as butter, the determination of volatile acidity is useful, since the lower fatty acids such as butyric acid are volatile in steam and this may be used for their separation and estimation. It is frequently useful to determine unsaponifiable matter, which consists of compounds such as sterols which remain after saponification of the acylglycerols and removal of the glycerol and soaps by means of solvents. The content of brassicasterol in the sterol fraction of fixed oils is limited by the Pharmacopoeia for some oils, e.g. maximum 0.3% for evening primrose oil and borage oil.

The acetyl value is the number of milligrams of potassium hydroxide required to neutralize the acetic acid freed by the hydrolysis of the acetylated fat or other substance. The oil is first acetylated with acetic anhydride, which combines with any free hydroxyl groups present, and the product is then isolated after thorough removal of acid resulting from the reagent; its saponification value is determined together with that of the original oil. The acetyl value is calculated from the difference between these two figures.

The hydroxyl value of a substance depends on the number of free hydroxyl groups present. It is expressed as the number of milligrams of potassium hydroxide required to neutralize the acid combined by acylation of the sample. Most fixed oils have low values, which arise from small quantities of sterols present; castor oil is an exception (minimum value 150), arising from the high proportion of ricinoleic acid present.

Under unsuitable storage conditions, such as exposure to light and air, fixed oils undergo secondary oxidation to give peroxides that then generate aldehydes and ketones. Such deterioration is detected by measurement of the peroxide value and anisidine value. The former is described by the Pharmacopoeia as ‘the number that expresses in milliequivalents of oxygen the quantity of peroxide contained in 1000 g of the substance, as determined by the prescribed methods’. These methods involve the liberation of iodine from a potassium iodide solution by the peroxides present in the sample and titration with 0.01 Msodium thiosulphate solution. For refined oils such as olive, borage, evening primrose and wheat-germ the typical value is 10; if these oils are to be used for parental dosage forms the maximum is 5. The maximum permissible value is higher for the virgin oils, e.g. olive = 20. Peroxide values are also used for fish oils, e.g. maximum value for farmed salmon oil, 5.0.

Anisidine values are determined photometrically (350 nm) and depend on the coloured complex produced by the interaction of p-anisidine (the methyl ether of p-aminophenol) with aldehydes and ketones. They are used principally for the evaluation of fish oils, including type-A cod-liver oil and farmed salmon oil (maximum 10).

Certain physical constants of fixed oils and fats are significant: specific gravity, melting point, refractive index and sometimes optical rotation (e.g. castor oil). Table 19.6 shows how chemical standards are related to chemical composition. The gas chromatographic separation and quantification of the acids produced by the hydrolysis of specific fixed oils is an official method for their identification and quality control; type chromatograms are included in the BP/EP. Such detailed analyses often eliminate the necessity of rountinely applying all the above quantitative standards. Some examples of this application are given for the oils in Table 16.4. Comments on the detection of adulterants in the more expensive oils can be found under individual headings.

Table 19.6 Numerical properties of fixed oils.