Chapter 2 Phytochemistry of Aptosimum procumbens 2.1 Introduction Aptosimum procumbens Burch (= A. depressum) of the tribe Aptosimae belongs to the Scropulariaceae sensu stricto family of the order Lamiales.1 Scrophulariaceae is one of the largest plant families and is comprised of about 190 genera and 4000 species. Plants from this family are mostly woody herbaceous shrubs and are found predominantly in the temperate regions of the world.2 They are distinguished from related families with relative ease, but many plants are assigned to this family because they lack distinguishing characteristics that would place them in the other specific families. Therefore, Scrophs share some of the characteristics of plants of related families and this may negate the possibility that Scrophulariaceae is a distinct clade. As a result of this, there are doubts as to whether the family is monophyletic or should rather be classified as polyphyletic or paraphyletic.1 Phytochemical investigations can provide a valuable input with regards to the chemotaxanomical studies of this family. A. procumbens (Fig. 1) is mainly referred to as “carpet flower” but shares a variety of vernacular names such as “brandbossie/blare”, “Karoo violet/flower” and “kankerbos” with other related species of Aptosimum. A common name is given to a plant based on certain characteristics of the plant. A. procumbens is a prostrate mat-forming species hence the name “carpet flower”. The plant has strong woody procumbent stems with short lateral, dense leafy and floriferous branches. Another characteristic feature is the violet trumpet-like flowers that bloom in the summer or after rainfall (hence the name “Karoo violet”). The dwarf shrub grows mostly in dry soil, on rocky terrain in full sun, in flood plains and is found in semi-arid regions of southern Africa (commonly in the Karoo region).3,4 6 http://www.museums.org.za/bio/plant/scrophulariaceae/aptosimum_procumbens.htm Figure 1: A. procumbens Aptosimum has been used as a traditional medicine in South Africa3,4 and in parts of Europe, such as Germany for the treatment of a variety of ailments such as water retention and micturition difficulties, diptheria, ringworm, impetigo lesions and krimpsiekte in sheep.5 It may be administered as a tea, gargle, decoction or paste depending on the disease it is being used to treat. When applied to the skin, the foliage may cause a blister suggestive of a burn and it is, therefore, also referred to as brandbossie. 6 A variety of Aptosimum species such as A. indivisum, A. decumbens, A. albomarginatum, A. angustifolium, A. spinescens, A. glandulosum, A. arenarium, A. sp, A. lineare and A. steingroeveri with similar physical and medicinal characteristics exist.4,6 In general, most of the species are used in wound healing or for the treatment of gastrointestinal disturbances. A. indivisum and A. sp are also referred to as the Karoo violet.3 A. sp is an unidentified species that has been used for snakebites and epilepsy. 7 A. albomarginatum is an abortifacient and is used to assist in the expulsion of the placenta and regulates the menstral cycle. A. spinescens also called “kankerbos”, has been used as treatment for cancer, hypertension and haemorrhoids and the leaves of A. decumbens may be chewed to improve memory.6 Schrophulariaceae is a rich source of iridoid glycosides, other compounds sometimes found in this family include orobanchins, triterpenoid saponins, cardiotonic glycosides and flavonoids.8 Plants containing iridoids generally have anti-inflammatory properties.9 Some of these plants, such as Devil’s claw (Harpagophytum procumbens), are used as traditional medicines10. 7 To date only two species of Aptosimum have been investigated namely, A. spinescens and A. indivisum. Eight lignans: sesamin (2.1), spinescin (2.2), piperitol (2.3), pinoresinol (2.4), pinoresinoldimethylether, pinoresinolmonomethylether, aptosimon (2.5) and aptosimol (2.6), have been isolated from A. spinescens.11 In a paper on the phytochemistry of A. indivisum the plant was wrongly identified as Craterocapsa tarsodes (Campanulaceae) (FR van Heerden, personal communication). Shanzhiside methyl ester (an iridoid) (2.7), verbascoside (2.8) and the flavanone pinocembrin 7-O-b- neohesperidoside (2.9) were isolated from A. indivisum.12 A. procumbens has been used as a traditional medicine in the Cape and the Karoo regions. In our laboratories the crude extract of A. procumbens showed moderate anticancer activity against three cell lines and we decided to perform a phytochemical investigation on the plant. O Ar1 1 2 2.1 Ar = Ar = 3,4-methylendioxyphenyl 2.2 Ar1 = 3,4 methylendioxyphenyl 2 Ar = 3,4-dimethoxyphenyl Ar 2 O 2.3 Ar1 = 3,4-methylendioxyphenyl Ar2 = 3-methoxy-4-hydroxyphenyl 2.4 Ar1 = Ar2 = 3-methoxy-4-hydroxyphenyl O R1 R2 2.5 Ar = 3,4 methylendioxyphenyl 1 2 R , R = O 2.6 Ar = 3,4-methylendioxyphenyl Ar O Ar 1 R = OH 2 R = H CO Me HO 2 H O H HO OH O HO O HO HO 2.7 8 O HO HO O O O OH O HO OH H3C O HO OH HO OH 2.8 HO HO O O O HO O H3C O HO OH O HO OH 2.9 2.2 Literature Review on Iridoids 2.2.1 Structural Classification Iridoids are monoterpenes and are found as natural constituents in a large number of plant families. These compounds have a characteristic cyclopenta[c]pyranoid skeleton also known as an iridane skeleton (cis-2-oxabicyclo [4, 3, 0 ]nonane) (2.10). They occur mainly in the form of glycosides. A methyl group (C-10) is found most commonly at the C-8 position and is rarely absent.13 The term iridoid was derived from the name s iridomyrecin, iridolacton and iridodial, compounds that are present in the defense secretions of certain ant species of the Iridomyrmex genus.14 11 6 4 5 3 7 O 8 9 1 10 2.10 9 There are four main categories of iridoids: agylcone iridoids, secoiridoids, bisiridoids and iridoid glycosides (the most abundant).13 Iridoids generally have nine carbons with a tenth carbon often bonded to C-4. This carbon may be a methyl group or it may form part of a carbonyl or secondary alcohol functional group. Structural variations and diversification of iridoid types are achieved by the introduction of additional carbons, functional groups and double bonds into the skeleton.13 Simple aglycone structures such as nepetalactone (2.11) are found in plants. This compound was isolated from Nepeta cataria (Lamiaceae), otherwise known as catnip, and is an (sometimes infamous) attractant of cats.15 However, most non-glycosidic iridoids form part of modified structures such as alkaloids, polycyclic compounds, polyesters and intramolecular ethers.13 A large portion of iridoid glycosides can be characterized as glucosides with a glycosidic linkage between the anomeric hydroxyl of D-glucose and the aglycone C-1 hydroxyl. A simple example of this is loganin (2.12), the biosynthetic precursor of many iridoids.16 In some rare cases, the glycosidic linkage may occur between the C-11 hydroxyl of the aglycone and anomeric carbon hydroxyl of D-glucose, as in ebuloside (2.13) isolated from the Caprifoliaceae family.13 The sugar type and complexity may also vary, for example catalpol (2.14) may have an additional rhamnosyl attached at C-6 to form 6-O- a-L-rhamnosylcatalpol (2.15). This is also an example of an iridoid that lacks an tenth carbon.17 CO2CH3 H H HO O O H H H 3C H3C O O-Glc 2.11 2.12 O-Glc RO H O O O O H H C 3 O OGlc OH 2.14 R = OH O 2.13 2.15 R = Rhamnosyl 10 Secoiridoids arise as a result of the cleavage of the 7,8 -bond of the cyclopentane ring. Secologanin (2.16) is a secoiridoid with a vinyl group at C-9. Various other structural modifications give rise to different secoiridoids. Aglycone secoiridoids do exist but are a rarity.18 O CO 2CH3 O O-Glc 2.16 Bisiridoids form as a result of dimerization of both iridoids and secoiridoids. Picconioside I (2.17) is a bisiridoid that consists loganin (part a) esterified with deoxyloganin (part b).19 Other structural variations include modification of the iridane skeleton and it’s functional groups. The methyl group (C-10) may be oxidized, giving rise to a secondary alcohol as in catapol (2.14). Epoxidation of the cyclopentane ring as in catapol (2.14) is another example of ring modification. Acetoxy groups may also be introduced as in lamioside (2.18). In deutzioside (2.19) the methyl group (C-10) is absent.13 CO2CH3 O O C O HO HO OH H OGlc O O O part a O AcO H H CH OGlc 3 O-Glc O-Glc part b 2.17 2.18 2.19 The presence of unsaturation as in aucubin (2.20) or monotropein (2.21) also leads to differentiation. The introduction of a hydroxyl at, or oxidation of C -6, C-7 and C-8 as in verbenalin (2.22), loganin (2.12) or monotropein (2.21) results in further structural diversification. 11 A notable structural variation is that certain iridoids possess carboxyl groups (C-11) and others do not. Iridoids can, therefore, also be classified as carboxylated and non- carboxylated. Moreover, they are derived from different biosynthetic precursors.20 COOH CO 2CH3 HO O H H H O O O HO H H H OH HO O-Glc O-Glc O-Glc 2.20 2.21 2.22 2.2.2 Biosynthesis of Iridoids The basic building blocks of terpenoids are C-5 isoprene units, in the form of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).