All about anthraquinones - part 1

Plant extracts containing anthraquinones are increasingly being used in cosmetics as well as in foods and pharmaceuticals. The first of this two-part paper investigates their sources and benefits

Aloe vera contains a large number of different anthra-quinones occuring as red or reddish yellow leaves

Plant extracts containing anthraquinones are increasingly being used in cosmetics as well as in foods and pharmaceuticals. The first of this two-part paper investigates their sources and benefits

Sweden has given the world many outstanding scientists: Arrhenius, Nobel, Scheele, Berzelius, Ångström, Rydberg, Celcius, Bøhlin and of course Carl von Linné, better known as Linnaeusl, who classified living species according to morphological properties, a system that is still used today.

Classification of living things (plants, animals, microorganisms) can also be achieved using the chemical principles - chemotaxonomy - in which the presence and distribution of various chemical compounds in plants serve as taxonomic evidence. Chemotaxonomy is still a young science; its foundations were only properly shaped only in 1987 (Wayne), and this part of botanical science is still very much in statu nascendi. To put it simply, particular plants produce particular terpenoids, steroids, alkaloids, anthraquinoids (another name for anthraquinones) and other products that are specific to a plant or genus of plants and not produced by others. The authors have previously presented papers here on steroids, flavonoids and alkaloids, and it is now the turn of anthraquinones.

Anthraquinones are condensed aromatic structures and the parent compound is anthracene. This highly crystalline aromatic hydrocarbon occurs in coal tar and melts at 218°C. It is insoluble in water but readily soluble in most organic solvents such as carbon disulfide, aliphatic hydrocarbons, alcohols, esters, aromatic benzene and halogenated solvents. Upon oxidation of anthracene with potassium dichromate/sulphuric acid, anthraquinone is obtained, a substance that is the parent compound for a large class of dyes and pigments.

Anthracene belongs to the family of polycyclic aromatic hydrocarbons and is not a friendly chemical. It is a severe irritant and sensitiser, both to the skin and eyes, and when taken orally it causes severe damage to the liver, kidneys and mucous membranes. Upon inhalation, severe lung damage will occur. On the other hand, anthraquinone (formal IUPAC name: 9,10-dioxoanthracene) is relatively non-toxic; the LD50-value (rat, oral) has been determined as 15g/kg body weight.

Coal tar is no longer used to produce anthraquinone. The anthraquinone skeleton can also be made by means of a Friedel-Crafts reaction of phthalic anhydride with something like catechol (1,2-dihydroxybenzene) using a strongly acidic catalyst such as anhydrous aluminium chloride or zinc chloride. The first reaction product is o-(3,4-dihydroxybenzoyl)-benzoic acid, and in a second Friedel Crafts reaction this produces a mixture of two anthraquinone dyes: alizarin and histazarin.

Alizarin forms orange-red needles, is poorly soluble in water and is easily soluble in ethanol and acetone. The value for ?max in alcohol is 435nm (log(e)=3,8). Histazarin forms yellow-brown needles, is insoluble in water and only poorly soluble in ethanol and acetone. Because of the presence of the two phenolic hydroxyl groups, both alizarin and histazarin are easily soluble in alkali.

Two monohydroxyanthraquin-ones are known, but they do not have any commercial value. Thirteen dihydroxyanthraquinones are possible and all are known. The most important ones after alizarin and histazarin are purpuroxanthin (1,3), anthrarufin (1,5), chrysazin (1,8) and anthraflavin (2,6). All of these occur naturally.

Alizarin is the main dye found in madder root (Rubia tinctorium, Rubia cordifolia). It is also known as Pigment Red 83 or CI 75330. The alizarin pigment is present in madder root as water soluble glycoside, while the aglycon is obtained by fermentation.

Madder extract is allowed in personal care and cosmetic products for hair dyeing without any restriction, and the same is true for Pigment Red 83, though funnily enough CI 75330 is not an allowed INCI name. As well as alizarin, madder contains another dye in high concentration, a phthalocyanin called purpurin, next to a variety of substituted dihydroxyanthraquinones, often as their methyl ethers, carboxaldehydes, or with a methyl group on the 2-position; 1,4-dihydroxy-2-methyl-anthraquinone is known as an effective termitifuge.

Alizarin itself also has 5-alpha-reductase inhibitor properties, making madder root functional to stimulate hair growth. It also occurs in noni (Morinda citrifolia), which is frequently recommended as a nutritional supplement for the treatment of leukaemia. At present the FDA does not accept these claims, although its study is ongoing.

Biosynthesis of anthraquinones

Malonyl-CoA plays an important role during the synthesis of steroids and flavonoids. It is also the starting point for the biosynthesis of anthraquinones via the formation and polymerisation of malonyl-CoA (MAT) units by polyketide synthases (PKS).

Malonyl-CoA is condensed with a keto synthase-chain length factor (KS-CLF) whereby an acetic acid moiety is released; the formation proceeds via an acyl carrier protein (ACP). Formation of the chain start by condensing additional malonyl-CoA units to the base unit under the influence of either an octaketide or a decaketide synthase. The highly complex biochemistry of these reactions has largely been unravelled (Tang, Lee & Khosla, 2004). Through the activity of particular enzymes such as octaketide kinase and decaketide synthase complex products are obtained and identified as octaketide and decaketide.

Ketides are a large group of molecules with highly diverse structural properties. A number of antibiotics and anti-tumour drugs are derived from polyketides, such as tetracycline and doxorubicin (O’Hagan, 1991), and it is worth noting that a small unit such as malonyl CoA using polyketide synthases results in tremendous biosynthetic variability. Micro-organisms produce these products and it is thought that microorganisms such as Actinomyces and Strep-tomyces species, that live in symbiosis with particular plants, offer protection to the plants while the host offers excellent hospitality to these microorganisms.

The geometry of the polyketides suggests the identity of particular molecules, such as naphthoquinones and tetracyclines as well as anthraquinones. Also, heterocyclic systems may be formed containing one or more oxygen atom. In all cases these products are classified as pharmacologically active.

The results are sometimes extremely challenging. Vertesy et al (Aventis, 2001) found that mumbaistatin, a complex 1,6-dihydroxy-7-carboxyanthra-quinone with a complex side chain on the 8-position produced by a Streptomyces species is the most powerful glucose-6-phosphatase translocase inhibitor known (IC50 = 5 nMole).

Using botanical extracts from plants that live in symbiosis with particular microorganisms, it is inevitable that these extracts also contain metabolites produced by these microorganisms. In a number of cases it has been shown that a significant part of the physiological activity of botanical extracts can be attributed to the work done by microorganisms. That doesn’t mean the production of these physiologically active products is the monopoly of microorganisms (bacteria, fungi). Particular plants are themselves able to produce them. Related to polyketide synthases are chalcone synthases and stilbene synthase. These enzymes catalyse the stepwise condensation between acyl co-enzyme A esters in the biosynthesis of flavonoids, stilbenes and other aromatic polyketides. All these enzymes have as a similar structural property a highly conserved cysteïne residue that is essential for the polyketide synthase activity. This is different for bacterial or fungal polyketide synthases where this cysteïne residue is not pronouncedly present.

The chemistry of flavonoids, steroids, anthraquinones, but also fatty acids, antibiotics and numerous other products looks very different, but the biochemical pathways are very similar. In all cases, the same building blocks are used - acetyl co-enzyme A or malonyl co-enzyme. In all cases large sets of enzymes are used and it is a miracle that the chemical sequences function; they have to be present at the right moment in the right concentration and are made to order. If we wanted to come close to the logistic abilities of a simple organism like a bacterium, we’d be living in a different world. The divined selectivity and specificity of the enzyme systems is a guarantee for ultimate chemical beauty. By all means, it is chemistry in optima forma, but the same chemistry is so often condemned.

Aloe vs rhubarb

It is interesting to observe that there is quite a lot of symmetry between the chemical composition of aloe and rhubarb species, particularly the anthraquinones present, despite the fact that the visuals of both plant groups have a very different chemotaxonomy.

More than 400 aloe species have been described, mainly occurring in Africa. The best known representatives are Aloe vera and Aloe ferox, which are of great cosmetic and medicinal value. Topically, aloe is used for burns, wound healing, psoriasis, sunburn, frostbite, inflammation, osteoarthritis and cold sores. It is also applied topically as an antiseptic and as a moisturiser. Aloe vera contains a large number of different anthraquinones, such as 1,8-dihydroxyanthraquinone (danthron). This occurs as red or reddish yellow needles or leaves and has a melting point of 193-195°C. Danthron is very soluble in alkaline conditions and is soluble in acetone, chloroform, ether and ethanol, but is practically insoluble in water. It has been widely used as a laxative, but in 1987 the FDA ordered its withdrawal from the market. It is currently used as an antioxidant in synthetic lubricants, in the synthesis of experimental anti-tumour agents and as a fungicide for control of powdery mildew.

Chrysophanol has haemostatic and bactericidal properties and is also found in the roots of regular rhubarb (Rheum rhabarbarum) and in significant levels in Chinese rhubarb (Rheum palmatum); more than 350 rhubarb species are known and most of these are edible. Chinese rhubarb, also called Da Huang, is used medicinally as a laxative, this activity being attributable to the presence of the anthraquinones. As well as chrysophanol, Chinese rhubarb contains rhein (antiviral, bactericide, viricide), emodin (anti-inflammatory, anti-tumour [breast], vasorelaxant, viricide) and aloe-emodin (anti-herpetic, anti-leukemic, viricide).

Chrysarobin is related to chrysophanol. This molecule lacks the carbonyl group on the 10-position of the anthracene system. Crude chrysarobin is also known as Bahia powder or Goa powder, and is obtained from the trunk of Andira araroba. In the British Pharmaceutical Codex it is named Araroba. Although Goa powder may act as a severe skin irritant, it has been demonstrated to be effective for the treatment of acne and eczema and is probably the most effective treatment known for psoriasis. However, when taken orally it readily causes gastroenteritis and colours the urine dark yellow.

Aloe and (Chinese) rhubarb also contain water soluble anthraquinones. These originate from oxidation of the methyl group in chrysophanol to the corresponding hydroxymethyl group (aloe-emodin). The carbonyl group on the 10-position is converted to a hemiacetal with rhamnose, and this product is named aloin. The consequence is that the carbon atom on the 10-position becomes chiral, and thus two optical isomers of aloin exist: aloin A and aloin B. The racemate is called barbaloin.

These glycosides are often subject to hydrolysis and subsequent oxidation, resulting in precipitation of water insoluble aloe-emodin. This procedure is also followed during processing of aloe vera gel. The gel is removed from the leaves and is obtained as a gel-like substance comparable to a high viscous carbomer gel. Upon oxidation of the whole gel with hydrogen peroxide the gel structure is destroyed and the water soluble anthraquinones are converted into water insoluble products that can be filtered off.

Barbaloin has antibacterial, antihistaminic and laxative properties. Upon oxidation of chrysophanol, the corresponding acid, chrysophanic acid, is obtained, which acts as a calcium antagonist and has anti-spasmodic properties.

Emodin (3-methyl-1,6,8-trihydroxyanthraquinone) has a wide variety of functionalites. It has bactericide and viricide properties, has powerful immune stimulating properties, is active against the cytomegalovirus (ED50=1.1 (g/ml) and has vasodilating properties. In high concentrations, however, emodin has cytotoxic properties.

Rhubarb has never really made it in cosmetics. This is a shame as rhubarb has a lot to offer cosmetics, and it’s not just limited to anthraquinones.

Recently, several anthraquinones have been isolated from the roots of Aloe berhana and Aloe graminicola (Dagne, Yenesew, 1994). It was shown that the anthraquinones are formed through two parallel routes of the polyketide pathway, differing in the way the octaketide chain is folded. This leads to two groups of anthraquinones: 1,8-dihydroxyanthaquinones and 1-hydroxy-8-methylanthraquinones. The latter are rarely encountered; examples are aloesaponarin I, aloesaponarin II and of course laccaic acid D.

Three other laccaic acids are known: A, B and C. Laccaic acid is permitted in personal care and cosmetic products without restrictions. It is used for semi-permanent hair dyeing and distinguishes itself by an excellent light and wash fastness.

Laccaic acid D is obtained from Laccifer lacca, an insect found in India and Thailand that lives in trees and covers itself with a waxy substance called shellac. Shellac is a hard, brittle wax composed mainly of long chain wax esters (>80%) and some wax acids (~12%). Interesting is the presence of an unusual fatty acid, aleuritic acid (9,10,16-trihydroxy-palmitic acid). This acid is an ideal precursor for synthetic ceramides such as Ceramide HO3 (Sederma). Aleuritic acid is also applied in fragrance preparations, mainly as its macrocyclic internal ester(s). Shellac wax has many and diverse applications, including citrus fruit coatings, fireworks, microprocessor technology etc. Shellac also contains a red dye identified as a representative of the group of 1-hydroxy-8-methylanthraquinones - laccaic acid D.

Another 1-hydroxy-8-methylanthraquinone derivative is carminic acid, a red dye also called cochenille (not to be confused with cochenille red, a red azo dye). The makeup of Cochenille is similar to laccaic acid D to such an extent that on the 2-position a glucosyl group is present, making the dye water soluble. This food grade dye (E120) is obtained from the insect Dactylopius coccus, cactus lice living on opuntia species. Only the females are used, the lifespan of the males being so short that they are useless for cochenille production. Peru is the major producer of cochenille with a market share of ~80%, although in recent times related insects producing cochenille have been cultivated in Poland and Armenia.