cardioactive drugs and other steroids

Chapter 23 Saponins, cardioactive drugs and other steroids

PENTACYCLIC TRITERPENOID SAPONINS 313

Plant materials containing saponins have long been used in many parts of the world for their detergent properties. For example, in Europe the root of Saponaria officinalis (Caryophyllaceae) and in South America the bark of Quillaja saponaria (Rosaceae). Such plants contain a high percentage of glycosides known as saponins (Latin sapo, soap) which are characterized by their property of producing a frothing aqueous solution. They also have haemolytic properties, and when injected into the blood stream, are highly toxic. The fact that a plant contains haemolytic substances is not proof that it contains saponins, and in the species examined by Wall (1961) only about half of those containing haemolytic substances actually contained saponins. When taken by mouth, saponins are comparatively harmless. Sarsaparilla, for example, is rich in saponins but is widely used in the preparation of non-alcoholic beverages.

Saponins have a high molecular weight and a high polarity and their isolation in a state of purity presents some difficulties. Often they occur as complex mixtures with the components differing only slightly from one another in the nature of the sugars present, or in the structure of the aglycone. Various chromatographic techniques have been employed for their isolation. As glycosides they are hydrolysed by acids to give an aglycone (sapogenin) and various sugars and related uronic acids. According to the structure of the aglycone or sapogenin, two kinds of saponin are recognized—the steroidal (commonly tetracyclic triterpenoids) and the pentacyclic triterpenoid types (see formulae below). Both of these have a glycosidal linkage at C-3 and have a common biogenetic origin via mevalonic acid and isoprenoid units.

A distinct subgroup of the steroidal saponins is that of the steroidal alkaloids which characterize many members of the Solanaceae. They possess a heterocyclic nitrogen-containing ring, giving the compounds basic properties (as an example see solasodine, Fig. 23.5).

STEROIDAL SAPONINS

The steroidal saponins are less widely distributed in nature than the pentacyclic triterpenoid type. Phytochemical surveys have shown their presence in many monocotyledonous families, particularly the Dioscoreaceae (e.g. Dioscorea spp.), Agavaceae (e.g. Agave and Yucca spp.) and Smilacaceae (Smilax spp.). In the dicotyledons the occurrence of diosgenin in fenugreek (Leguminosae) and of steroidal alkaloids in Solanum (Solanaceae) is of potential importance. Some species of Strophanthus and Digitalis contain both steroidal saponins and cardiac glycosides (q.v.). Examples of saponins and their constituent sugars are given in Table 23.1.

Table 23.1 Examples of steroidal saponins.

Steroidal saponin	Sugar components	Occurrence
Sarsaponin (Parillin)	3 glucose, 1 rhamnose	Smilax spp.
Digitonin	2 glucose, 2 galactose, 1 xylose	Seeds of Digitalis purpurea and D. lanata
Gitonin	1 glucose, 2 galactose, 1 xylose	Seeds and leaves of D. purpurea and seeds of D. lanata
Dioscin	1 glucose, 2 rhamnose	Dioscorea spp.

Steroidal saponins are of great pharmaceutical importance because of their relationship to compounds such as the sex hormones, cortisone, diuretic steroids, vitamin D and the cardiac glycosides. Some are used as starting materials for the synthesis of these compounds. Diosgenin is the principal sapogenin used by industry but most yams, from which it is isolated, contain a mixture of sapogenins in the glycosidic form.

As with cardiac glycosides, the stereochemistry of the molecule is of some importance, although not so much so for cortisone manufacture. Natural sapogenins differ only in their configuration at carbon atoms 3, 5 and 25, and in the spirostane series the orientation at C-22 need not be specified (cf. steroidal alkaloids). Mixtures of the C-25 epimers—for example, diosgenin (Δ⁵,25α-spirosten-3β-ol) and yamogenin (Δ⁵,25β- spirosten-3β-ol)—are of normal occurrence and their ratio, one to the other, is dependent upon factors such as morphological part and stage of development of the plant. In some instances in the plant, the side-chain which forms ring F of the sapogenin is kept open by glycoside formation as in the bisdesmosidic saponin sarsaparilloside of Smilax aristolochiaefolia.

BIOGENESIS OF STEROIDAL SAPONINS

Steroidal saponins arise via the mevalonic acid pathway; the preliminary stages have been discussed in Chapter 18. A scheme for the subsequent cyclization of squalene to give cholesterol is illustrated in Fig. 23.1. Cholesterol, the wide distribution of which in plants has only relatively recently been shown, can be incorporated into a number of C₂₇ sapogenins without side-chain cleavage (Fig. 23.2), although it is not necessarily an obligatory precursor. Extensive investigations involving whole plants, homogenates and cell cultures have been performed to elucidate these detailed pathways, including the origin of the 25-epimers (e.g. diosgenin and yamogenin).

Fig. 23.1 Possible route for the formation of cholesterol in higher plants and algae.

Fig. 23.2 Some plant metabolites of cholesterol.

As early as 1947 Marker and Lopez had postulated that steroidal saponins exist in plants in a form where the side-chain is held open by glycoside formation. However, direct evidence for the natural occurrence of these compounds was not forthcoming for another 20 years. It has been shown that such open-chain saponins are, like the more common ones, formed from cholesterol. In Dioscorea homogenates one such compound has been converted to dioscin (a diosgenin glycoside) (Fig. 23.3).

Fig. 23.3 Formation and metabolism of an open-chain saponin in Dioscorea.

NATURAL STEROIDS FOR THE PRODUCTION OF PHARMACEUTICALS

Although total synthesis of some medicinal steroids is employed commercially, there is also a great demand for natural products which will serve as starting materials for their partial synthesis.

As indicated in Fig. 23.4, which illustrates the range of steroids required medicinally, cortisone and its derivatives are 11-oxosteroids, whereas the sex hormones, including the oral contraceptives, and the diuretic steroids have no oxygen substitution in the C-ring. Fig. 23.5 shows some of the more important natural derivatives which are available in sufficient quantity for synthetic purposes. Hecogenin with C-ring substitution provides a practical starting material for the synthesis of the corticosteroids, whereas diosgenin is suitable for the manufacture of oral contraceptives and the sex hormones. Diosgenin, however, can also be used for corticosteroid synthesis by the employment, at a suitable stage in the synthesis, of a microbiological fermentation to introduce oxygen into the 11α-position of the pregnene nucleus.

Fig. 23.4 Examples of structures of therapeutically active steroids.

Fig. 23.5 Some naturally occurring steroids.

Efforts are constantly being made to discover new high-yielding strains of plants and to assure a regular supply of raw material by the cultivation of good-quality plants. Hardman in a review on steroids (Planta Med., 1987, 53, 233) recorded that, annually, the American Chemical Abstracts contained some 3000 references pertinent to plant steroids or related compounds. Some of the better-known examples of steroidal sapogenins and their sources are given in Table 23.2. (For a review, tabulating over 200 sapogenins, see A. V. Patel et al., Fitoterapia, 1987, 58, 67.)

Table 23.2 Some steroidal sapogenins and their sources.

Sapogenin	Species	Location
Diosgenin	Dioscorea sylvatica	Transvaal and Natal
	D. mexicana and D. composita	Mexico and Central America
	D. collettii, D. pathaica and D. nipponica	China
	D. floribunda	Guatemala and cultivated in India
	D. deltoidea and D. prazeri	India
	D. tokoro	Japan
	Costus speciosus	India
	Kallstroemia pubescens	Tropical America; introduced into West Brazil
	Trillium spp.	North America
	Trigonella foenum-graecum	India, Egypt, Morocco
Hecogenin	Agave sisalana	Subtropical America and cultivated in Kenya for sisal and saponin
	A. rigida	Mexico
	Hechtia texensis	Central America
Sarsapogenin	Yucca spp., Smilax spp.	Central America
Sarmentogenin	Strophanthus spp.	Africa

Dioscorea species

Tubers of many of the dioscoreas (yams) have long been used for food, as they are rich in starch. In addition to starch, some species contain steroidal saponins, others alkaloids. From a suitable source the sapogenins are isolated by acid hydrolysis of the saponin. Previous fermentation of the material for some 4–10 days often gives a better yield. The water-insoluble sapogenin is then extracted with a suitable organic solvent. Both wild and cultivated plants are used. Cultivation requires attention to correct soil and drainage, support for the vines and freedom from weeds, virus, fungus and insect attack. According to the species, the tubers reach maturity in 3–5 years and on average, yield 1–8% of total sapogenin.

Until 1970 diosgenin isolated from the Mexican yam was the sole source for steroidal contraceptive manufacture. With the nationalization of the Mexican industry, however, prices were increased to such an extent that manufacturers switched to hecogenin for corticosteroids, to other sources of diosgenin and to the use of the steroidal alkaloids of Solanum species. Total synthesis also became economically feasible and is now much used. More recently, the economics of steroid production have again changed in that China is now exporting large quantities of diosgenin; it is of high quality, being free of the 25β-isomer yamogenin, although this is of no commercial significance, and is reasonably priced. Three of the many Dioscorea spp. found in China and used commercially are given in Table 23.2; the tubers of these yield 2% of diosgenin, with the average content of diosgenin for the main areas of production (Yunnan Province and south of the Yangtze River) being 1%.

Sisal

Hecogenin is obtained commercially as the acetate in about 0.01% yield from sisal leaves (Agave sisalana). In East Africa, from leaf ‘waste’ stripped from the leaves during removal of the fibre, a hecogenin-containing ‘sisal concentrate’ is produced. From this the ‘juice’ is separated and allowed to ferment for 7 days. The sludge produced contains about 80% of the hecogenin originally present in the leaves; steam at 1380 kPa pressure is employed to complete the hydrolysis of the original glycosides. By filtration and drying a concentrate containing about 12% hecogenin and varying amounts of other sapogenins is produced. This crude material is shipped for further processing and cortisone manufacture. Hecogenin is also produced in Israel and China. A number of new steroidal saponins have been isolated from the dried fermented residues of Chinese A. sisalana forma Dong No. 1 (see Yi Ding et al., Chem. Pharm. Bull., 1993, 41, 557).

A survey of 34 species of Agave by Blunden and colleagues in 1978 showed that the extracts of most yielded steroidal sapogenins. Previously it had been shown that certain commercial samples of crude sapogenins from A. sisalana also contained the dihydroxy steroid rockogenin, sometimes in appreciable quantity; this compound appears to be an artefact formed during processing and should be avoided. A dihydroxyspirostane, barbourgenin, has been described (G. Blunden et al., J. Nat. Prod., 1986, 49, 687). Agave hybrids with a high hecogenin content and relatively free of tigogenin, with which it is usually associated, have been developed. Gbolade et al. (Fitoterapia, 1992, 63, 45) reported on factors (season, geographical location) affecting steroidal sapogenin levels in Nigerian Agave and Furcraea species.

Another genus of the Agavaceae which has been systematically studied for the presence of steroidal compounds is Cordyline in which many sapogenins, including 1,3-dihydroxysapogenins, have been detected.

FENUGREEK

Although included in this section as a potential industrial source of diosgenin, the seeds of Trigonella foenum-graecum L. (Leguminosae) are also described in the BP and EP. However their principal current use is as a spice; India, Morocco and Egypt among others being important producers.

The very hard seeds have a strong characteristic odour and are irregularly rhomboidal and oblong or square in outline. They are somewhat flattened and are divided into two unequal parts by a groove in the widest surfaces. Their shape and size, and position of the embryo and hilum are shown in Fig. 23.6. The BP/EP describes the seeds as brown to reddish-brown but in commerce olive-green or yellow-brown samples are oftenencountered. Microscopically the testa and hypodermis are characteristic and the mucilage-containing endosperm enables the swelling index (q.v.) of the seeds (not less than six) to be used as a test of purity.

Fig. 23.6 Commercial fenugreek seed. A, Morocco, Israel; B, Ethiopia; C, India, Pakistan; D, transverse section of a seed. c, Cotyledons; e, endosperm; f, furrow; hm, hilum and micropyle region; r, radicle; t, testa.

(After Fazli and Hardman, Trop. Sci., 1968 10, 66.)

Fenugreek contains the simple pyridine-type alkaloid trigonelline; this base, also reported in garden peas, hemp seed, coffee and many other products was first isolated and described by Jahns in 1885. Today it serves as the reference substance for the BP/EP TLC identification test for the drug. Pharmaceutical manufacturing interest lies in a number of steroidal sapogenins, particularly diosgenin which is contained in the oily embryo. Fazli and Hardman investigated a number of commercial samples of seed as possible commercial sources of diosgenin (see reference in Fig. 23.6) and reported contents of 0.8–2.2% expressed on a moisture-free basis. In 1986, Gupta and colleagues isolated a series of furostanol glycosides (F-ring opened) named trigofoenocides A–G. In a series of papers, further furostanol glycosides designated trigoneosides Ia, Ib–Xlla have been reported in Egyptian seeds (T. Murakami et al., Chem. Pharm. Bull., 2000, 48, 994). As with dioscoreas, the yield of diosgenin is increased by fermentation of the seeds prior to acid hydrolysis. Although the diosgenin yield is lower than that of the dioscoreas, fenugreek is an annual plant which will also give fixed oil, mucilage, flavouring extracts and high-protein fodder as side-products. A number of Hardman’s registered varieties have been subjected to field trials in the UK. However, as long as other cheap sources of diosgenin are available commercially, fenugreek must be regarded as a fall-back source for this sapogenin.

A non-essential amino acid, 4-hydroxyisoleucine, first identified by Fowden et al. in 1973, constitutes up to 80% of the free amino acid composition of fenugreek seeds and has been shown to possess insulin-stimulating properties both in vitro and in vivo (C. Haefelé et al., Phytochemistry, 1997, 44, 563). Some 39 components of the volatile oil fraction have been identified.

Based on mucilage content, the BP/EP sets a swelling index of not less than 6.0 for the powdered seeds.

In addition to its use as a spice and potential source of diosgenin fenugreek is widely employed in traditional systems of medicine. Its antidiabetic, cholesterol-lowering, anti-inflammatory, antipyretic, antiulcer and anticancer properties have been demonstrated.

Further reading

Petropoulos GJ, Hardman R, editors (series ed). Fenugreek: The genus Trigonella. CRC Press, Taylor and Francis Group. Boca Raton, FL, 2002;200 pp. 646 refs

Solanum species

This large genus (over 1000 spp.) is noted for the production of C₂₇ steroidal alkaloids in many species. Some of these alkaloids are the nitrogen analogues of the C₂₇ sapogenins (e.g. solasodine and diosgenin: Fig. 23.5). Another series of C₂₇ compounds contain a tertiary nitrogen in a condensed ring system (e.g. solanidine; Fig. 23.2). These compounds can also be employed in the partial synthesis of steroidal drugs, and a number of companies have devoted considerable attention to commercial production. Species so exploited are Solanum laciniatum, S. khasianum (a nearly spineless variety has been produced) and S. aviculare; trials on the production of S. marginatum have been conducted in South America. Zenk and colleagues have assayed over 250 spp. of Solanum for solasodine. A number of new glycosides have been isolated from S. dulcamara leaves and include two based on tigogenin and two on soladulcidine.

The steroidal alkaloids were reviewed in 1993 by Atta-ur-Rahman and M. I. Choudhary (Methods in Plant Biochemistry (ed. P. G. Waterman) Vol. 8. Academic Press, London, p. 451).

Soya bean sterols

The soya (soy, soja) plant, Glycine max (G. soya) (Leguminosae) is extensively cultivated for its seeds, which are rich in oil and protein. The seeds also contain appreciable quantities of the phytosterols stigmasterol and sitosterol (Fig. 23.5). Although not sapogenins, they are included here because they are now used extensively for steroid synthesis. They are obtained as byproducts of soap-making, being components of the unsaponifiable matter of the fixed oil. Pure stigmasterol, with its unsaturated side-chain is amenable to chemical conversion to suitable starting materials and can replace diosgenin. But it was more recently that sitosterol, the saturated side-chain of which could not be removed chemically without ring fragmentation, became commercially useful as the result of the discovery of a suitable microbiological side-chain removal. Both phytosterols are now processed by microorganisms. Similar phytosterols are found in other products—for example, cotton-seed oil, tall-oil (from the wood-pulp industry) and sugarcane wax.

For details of the soya isoflavones and their dietary importance, see Chapter 32: The plant nutraceuticals.

Sarsaparilla root

Sarsaparilla consists of the dried roots and sometimes also of the rhizomes of species of Smilax (Liliaceae, modern authors, Smilacaceae). The determination of the exact geographic and botanical sources of the numerous varieties which have from time to time been imported has been a matter of some difficulty (see Table 23.3).

Table 23.3 Varieties of sarsaparilla.

Variety and geographic source	Synonyms	Botanical source
Mexican (Southern Mexico, Guatemala, British Honduras)	Vera Cruz or Grey	Smilax aristolochiaefolia
Honduras (Guatemala, British Honduras, Honduras, cultivated in Jamaica)	Brown	S. regelii
Ecuadorian and Peruvian	Guayaquil	S. febrifuga
Central American	Costa Rica or ‘Jamaican’	Undetermined spp.

The plants produce numerous roots, 3 m or so long, which are attached to a short rhizome. The roots are cut, sufficient, however, remaining in the ground for the plant to resume its growth. Sometimes the rhizomes as well as the roots are collected. After drying in the sun the drug is made into bundles and the bundles into bales.

Sarsaparilla is imported in large bales bound with wire. Each bale usually contains numerous bundles of approximately uniform size. These consist of long roots, with or without pieces of rhizome and aerial stems. The commercial varieties (Table 23.4) differ from one another in colour, ridges and furrows; in the presence or absence of rhizome and aerial stems; in the relative proportions of cortex, wood and pith, as seen in transverse section; in their microscopical structure. The drug is nearly odourless but has a somewhat sweetish and acrid taste. Owing to the presence of saponins, aqueous extractives froth readily.

Table 23.4 Macroscopical characters of sarsaparilla.

Much chemical work has been done on sarsaparillas without proper botanical identification of the material. Different species contain one or more steroidal saponins. Two isomeric genins are known: smilagenin and sarsasapogenin. These differ only in their configuration at C-25 and correspond to the reduced forms of diosgenin and yamogenin respectively. The principal crystalline glycoside of Smilax aristolochiaefolia is parillin (sarsasaponin, sarsasaponoside); it was first isolated from a sample of Jamaica sarsaparilla in 1913 by Power and Salway. On hydrolysis it gives sarsasapogenin, three molecules of glucose and one of rhamnose. Sarsaparilloside, contained in the same species, is a bisdesmosidic saponin (i.e. it possesses two distinct glycosyl groupings, in this case at C-3 and C-26) and represents the parillin molecule with an opened F-ring stabilized by glucosylation.

Uses

Sarsaparilla formerly enjoyed a high reputation in the treatment of syphilis, rheumatism and certain skin diseases. It is included in the BHP (1960) where it is indicated in the treatment of psoriasis and eczema, and for rheumatism and rheumatoid arthritis. Its action would appear to arise from the steroid content of the roots. Sarsaparilla is widely used as a vehicle, and large quantities are employed in the manufacture of non-alcoholic drinks. The genins are used in the partial synthesis of cortisone and other steroids.

Black Cohosh

Black Cohosh, Cimicifuga BHP 1983, is the dried rhizome and roots of Actea racemosa L. syn. Cimicifuga racemosa [L.], Nutt., family Ranunculaceae. This perennial plant is native to Canada and the northern American states and was well-known to the native Americans of these areas. The drug contains substances with endocrine activity and extracts have been widely employed in herbal medicine to treat menopausal and other female disorders, as well as various rheumatic conditions. However, in the 1990s doubts arose over its safety following reports concerning its hepatotoxicity; restrictions on its use have now been applied in a number of countries.

The rhizomes contain a number of triterpenoid glycosides, including actein, 23-epi-26-deoxyactein and cimiracemoside C. Cimicifugoside M and cimifugin can specifically serve as indicators for species identification (K. He et al., Planta Med., 2002, 66, 635). Phenyl propanoside esters are also present including cimiracemates A–D together with ferulic acid, isoferulic acid and methyl caffeate (S.-N. Chen et al., Phytochemistry, 2002, 61, 609). For other reports, see M. Nishida et al., Chem. Pharm. Bull., 2003, 51, 1215; H. Yoshitsu et al., Chem. Pharm. Bull., 2006, 54, 1322.

BUTCHER’S BROOM

Butcher’s broom, Ruscus aculeatus L. family Liliaceae, is a perennial, rigid, dark green much-branched bush, 2–3 feet in height. The leaf-like structures, twisted at the base and terminating in a sharp point, are actually cladodes—flattened stems or internodes that resemble and function as leaves. Small white flowers that arise in the axils of scarious bracts are followed by red berries, which appear situated on the surface of the cladodes.

The species is found in woods and dry places, extending across southern Europe to the Caucasus, and northward to Belgium. It is common in some southern areas of England.

The BP/EP drug consists of the dried, whole or broken roots and rhizomes of the plant. These are collected in autumn and dried to give yellow, knotty pieces of rhizome up to 10 cm in length, showing stem scars on the upper surface and roots and root-scars on the lower surface. A very hard, central cylinder can easily be removed from the outer layer.

Microscopical characteristics, as seen in the powder, include cells with thickened beaded walls with large oval pits, thin-walled parenchyma containing calcium oxalate, thick-walled fibres, and small vessels.

The rhizomes of this plant contain saponins related to those of Dioscorea; thus, one sapogenin is 1β-hydroxydiosgenin (ruscogenin). The plant glycosides involve up to three sugars attached at the C-1 hydroxyl with glucose terminating an uncyclized side-chain at C-26 (for detailed structures see Bombardelli et al., Fitoterapia, 1972, 43, 3). In a series of publications on Ruscus aculeatus M. Yoshikawa et al. have described many more saponins of both the spirostanol and furostanol series and recently, a new saponin which is unique in having a diglucoside unit at C-23 of the spirostanol skeleton (Phytochemistry, 1999, 51, 689). Both the alcoholic extract of the roots and the ruscogenins themselves have anti-inflammatory activity, produce diminished capillary permeability and exert a vasoconstrictor effect in the peripheral blood vessels. On the continent of Europe ointments and suppositories containing the active constituents are available for the treatment of conditions responding to the above effects.

The BP/EP TLC test for identity uses stigmasterol and ruscogenins as reference substances and a vanillin reagent for their visualization. A minimum of 1.0% total sapogenins, expressed as ruscogenins (mixture of neoruscogenin and ruscogenin) is specified; liquid chromatography is used for the assay.

Allied species

Various ruscogenins and a major new saponin have been detected in the rhizomes of Ruscus colchicus and R. hypoglossum (E. de Combarieu et al., Fitoterapia, 2002, 73, 583; 2003, 74, 423, corrigendum).

ELEUTHEROCOCCUS

The drug Siberian Ginseng (BP/EP, BHP) consists of the dried, whole or cut organs of Eleutherococcus senticosus Maxim. [Acanthopanax senticosus (Rupr. et Maxim.) Harms], family Araliaceae. The plant is native to China and is now cultivated there and in Russia, Japan and Korea.

Characters

The pale brown, uneven rhizomes, up to 4 cm in diameter, bear the scars of aerial stems and on the lower surface are roots and root scars. The fracture is fibrous and the internal surface light brown to pale yellow. The cylindrical roots are of variable length, up to about 1 cm in diameter, knotty and somewhat branched. When dry the bark is not easily removed from the underlying xylem; commercially it may be removed at harvest and both wood and bark used. Features of the powder include lignified fibres, reticulate and border-pitted vessels, parenchymatous cells containing cluster crystals of calcium oxalate and secretory canals with brown contents. Odour faintly aromatic; taste bitter and persistent.

Constituents

The rhizomes and roots contain a number of constituents termed eleutherocides (A to G; M). These, however, are not all of the same chemical group but include the phenyl propane glycosides eleutheroside B (syringin) and its aglycone sinapyl alcohol; also the lignans (−)-syringaresinol diglucoside (E) and its stereoisomer (D) (formulae Table 21.7), together with caffeic acid and its ethyl ester, chlorogenicacid and coniferaldehyde. Coumarins include coumarin, isofraxidin 7-glucoside (B₁) and sesamin (B₄). A group of heteroglycans (eleutherans A to G) have been studied for their hypoglycaemic activity. Other constituents include hedera-saponin (M), daucosterol (A) and volatile oil, about 0.8%.

The BP/EP requires a minimum content of 0.8% for the sum of eleutheroside B and eleutheroside E determined by liquid chromatography using UV spectrophotometric absorption at 220 nm and measurement of peak areas. These eleutherosides are also characterized by the TLC test for identity.

DNA analysis has been used to authenticate Japanese and Chinese commercial samples of the drug (T. Maruyama et al., Planta Medica, 2008, 74, 787).

Uses

Eleutherococcus has been used in Chinese medicine since antiquity for the treatment of rheumatoid complaints and for its revitalization properties. For many years, its adaptogenic qualities have been utilized in former USSR countries and now in W. Europe it is employed as a tonic in states of fatigue.

Further reading

Davydov M, Krikorian AD. Eleutherococcus senticosus (Rupr. and Maxim.) (Araliaceae) as an adaptogen: a closer look. J. Ethnopharmacology. 2000;72:345-393. An extensive discussion including chemical constituents, numerous formulae, over 200 refs

GINSENG

For some 2000 years the roots of Panax ginseng C. A. Meyer (Araliaceae) have held an honoured place in Chinese medicine. Today it is a product of world-wide usage. Production is principally confined to China, Korea and Siberia, although it is cultivated commercially on a small scale in Holland, England, Germany and France (Champagne district).

The most expensive ginseng is that derived from Korean root. The plant, about 50 cm tall with a crown of dark green verticillate leaves and small green flowers giving rise to clusters of bright red berries, is cultivated under thatched covers and harvested when 6 years old. Sun-drying of the root, after removal of the outer layers, produces white ginseng, whereas the red ginseng is obtained by first steaming the root, followed by artificial drying and then sun-drying. Rootlets are numerous on the lower surface of white ginseng but normally absent from red ginseng. The roots are graded and packed. Nineteenth-century descriptions record the care then taken with the preparation, silk, cotton and paper wrappings being used according to the quality of the drug; the wrapped roots were finally stored in containers with quicklime. Small roots are processed separately and form a separate article of commerce. The BP/EP recognizes both red and white ginseng.

The scraping of the roots before drying would appear to be disadvantageous because histochemical tests and GLC analysis show the active saponins to be located outside the root cambium.

Constituents

P. ginseng roots have been thoroughly studied by modern methods of analysis and, of the many compounds isolated, the medicinal activity appears to reside largely in a number of dammarane-type saponins termed ginsenosides by Japanese workers and panaxosides by Russian workers. These two series of compounds, all now generally termed ginsenosides, are glycosides respectively derived from the diol 20(S)-protopanaxadiol and the triol 20(S)-protopanaxatriol. Examples of the former are ginsenosides R_b1, R_b2 and R_b4 and of the latter ginsenosides (panaoxosides) R_e, R_f, R_g1, R_g2 (see Fig. 23.7). Acid hydrolysis of these saponins involves ring closure of the aglycone giving either panaxadiol or panaxatriol (Fig. 23.7). Some 30 ginsenosides have been named, although not all fit into the above scheme, e.g. ginsenoside R_o is an oleanolic acid derivative. Glucose is the principal sugar involved with some input of arabinose and rhamnose.

Fig. 23.7 Steroids associated with ginseng.

The BP/EP specifies a minimum of 0.40% for the sum of ginsenosides R_g1 and R_b1; this is determined by liquid chromatography of a methanolic extract using a reference solution containing the two ginsenosides to he assayed, with absorption measurements at 203 nm. TLC is used as a test for identity and to exclude substitution with P. quinquifolium, which contains no ginsenoside Rf.

Two other groups of compounds present in the root which have known therapeutic activity are high molecular weight polysaccharides (glycans) and acetylenic compounds. The glycans of P. ginseng have been named panaxans (A–U); panaxans A and B have been shown to be constituted mainly of α-(1 → 6) linked D-glucopyranose units with C-3 branching and a small component of peptide. (For the isolation of other polysaccharides termed ginsenans see M. Tomoda et al., Biol. Pharm. Bull., 1993, 16, 1087.) Those glycans tested have hypoglycaemic, antiulcer and immunological properties. One acidic polysaccharide MW 150000, originally isolated in 1993, and composed of 3.7% protein, 47.1% hexoses and 43.1% galacturonic acid, has antineoplastic immuno-stimulant properties.

A considerable number of mainly C₁₇, but also C₁₄, polyacetylenic alcohols have been isolated from the roots in recent years and are typified by panaxynol and panaxydol (Fig. 23.8). These compounds have been shown to have antitumour properties and Japanese patents exist for their isolation and derivatization. The cytotoxic activity of the C₁₇-polyacetylenes against leukaemia cells has been shown to be almost 20 times greater than that for the C₁₄-compounds (Y. Fujimoto et al., Phytochemistry, 1992, 31, 3499). K. Hirakura et al. have characterized a series of polyacetylenes named ginsenoynes A–K (see Phytochemistry, 1992, 31, 899 and references cited therein) and subsequently (ibid., 1994, 35, 963) three new linoleoylated polyacetylenes.