Fitoterapia 79 (2008) 401–418www.elsevier.com/locate/fitote

Review

Biology and chemistry of Ginkgo biloba

Bikram Singh ⁎, Pushpinder Kaur, Gopichand, R.D. Singh, P.S. Ahuja

Institute of Himalayan Bioresource Technology, Palampur, HP — 176 061, India

Received 7 January 2008; accepted 12 May 2008Available online 27 June 2008

Abstract

Ginkgo biloba has been existing on earth since 200 million years and is considered as a “living fossil”. It is among the most soldmedicinal plants in the world. A number of secondary metabolites representing terpenoids, polyphenols, allyl phenols, organicacids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids have been isolated from the plant. However, the mainbioactive constituents are terpene trilactones and flavonoid glycosides which are considered responsible for the pharmacologicalactivities of its standardized leaf extract. Scattered information is available on the extraction and analysis of these pharma-cologically important constituents which have been compiled in the present review.© 2008 Elsevier B.V. All rights reserved.

Keywords: Ginkgo biloba; Biology; Chemistry

Contents

1. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4022. Nomenclature and systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4023. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4034. Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4035. Sexual reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4036. Seed dispersal and establishment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4047. Tree architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4048. Seed production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4059. Leaf production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40510. Cultivation and propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40511. Pharmacological importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40912. Economical importance of the plant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40913. Secondary metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412

13.1. Flavonoid glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416

⁎ Corresponding author. Fax: +91 1894 230433.E-mail address: bikram_npp@rediffmail.com (B. Singh).

0367-326X/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2008.05.007

402 B. Singh et al. / Fitoterapia 79 (2008) 401–418

1. History

Ginkgo biloba L.(Syn.: Salisburia adiantifolia, Salisburia biloba Hoffmag-Common names: maidenhair-tree,ginkgo), has identity as a valuable plant for mankind since more than 2000 years and is considered a “living fossil”(Fig. 1). Though its natural habitat is in China, Japan and Korea, its place of origin is believed to be remotemountainous valleys of Zhejiang province of eastern China [1,2]. Up to 350 years ago, knowledge about this plantresource was limited to China. More than 100 plants of G. biloba ageing over 1000 years still exist around temples inChina [2] as its nuts are used in worship. Ginkgo probably originated about 200 million years ago, though itsswimming sperm (spermatozoids) were discovered hundred years ago [2].

Currently, G. biloba does not have any close relative in the plant kingdom. Thus, it is classified in a separatedivision, the Ginkgophyta. This taxon is distinguished from the Coniferophyta (Conifers) on the basis of itsreproductive structures, its multiflagellated sperm cells, and from the Cycadophyta (Cycads) owing to its vegetativeanatomy [3]. Molecular characterization of the genome has indicated that Ginkgo is distant from conifers and rathermuch closer to the Cycads [2]. The uncertainty has recently been resolved in Henan Province, China, by the discoveryof fossils belonging to the Middle Jurassic period (180 million years ago) with Ginkgo like ovule bearing organs [4].

Ginkgo yimaensis is a new species that differs from G. biloba in having more highly dissected leaves and muchsmaller ovules clustered attached on branched peduncles. Other extinct speciesG. adenoids had an extensive distributionin the northern hemisphere from the lower Cretaceous through the Pliocene and many researchers consider it to be theancestor of G. biloba because it had a similar type of leaf and ovule structure. About 98–65 mya, during the upperCretaceous period the fossil record for Ginkgo shows a decline in diversity and distribution, particularly due to loweringthe temperature. The genus disappeared from polar areas through the end of the Miocene. By the end of the Pliocene, astemperature declined and the rainfall slightly shifted from summer-wet to summer-dry, the genus Ginkgo was nottraceable in Europe. From south western Japan, the G. biloba was reported in Pleistocene record. Based on leafcharacteristic, these fossils were classified and admitted as an extinct species G. biloba [2]. In terms of the reproductiveorgans, the trend within the genus Ginkgo appears to be on the decline in the number of ovules with a contemporaneousincrease in the size of the ovules, loss of the pedicels resulting in sessile ovules connected directly to the peduncle [4–6].

2. Nomenclature and systematics

Linnaeus (1771) was the first pioneer who described G. biloba. Some other names like S. adiantifolia Smith andPterophyllus salisburiensis Nelson are not considered its valid synonyms as per the rules of priority. The Germanname is not in practical use. The English term “Maidenhair tree” is based on the resemblance to the foliage of the“Maidenhair fern” (Adiantum). In Japan, it is known by “Ginkyo”. In France, it is known as “L'arbre aux Quaranteecus” and “Noyer Du Japon”. Earlier the Ginkgo spp. was included in Taxaceae. In fact this family includedPodocarpaceae and Cepholotaxaceae and has always been considered very artificial. A discovery in 1895 revealed that

Fig. 1. Ginkgo biloba L.

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Ginkgo possesses multicoated spermatozoid, and it formed the basis of Engler's classification of a particular family andclass of Ginkgoopsida [7] which dates back as old as the lower Jurassic [8]. This group reaching at its highest positionin upper Jurassic and bottom in Cretaceous period had also disappeared rapidly before the end of the Cretaceous. In theOligocene time, only two genera out of the 19 genera comprising about 60 species of the family became extinct.Currently, these spp. are non-traceable from all but one continent, in which only one single species exists owing tonatural or cultural reasons [9]. Thus, G. biloba is the prime botanical example of a “living fossil”. This term was earlierused by Darwin for the King Crab (Limulus sp.). The systematic classification of G. biloba is as follows [10].

Division: Spermatophyta

1. Sub-division: Coniferophytinai) Class: Ginkgoopsidaii) Class: Pinopsida (with three sub-classes).

2. Sub-division: Cycadophytinai) Class: Lyginopteridopsida (Seed fern)ii) Class: Cycadopsida (Cycadaceae a. o.)iii) Class: Pinopsida (Only known in fossil form)iv) Class: Gnetopsida (Ephedra, Welwitschia, Gnetum)

3. Sub-division: Magnoliophytina (Angiospermae).

3. Distribution

G. biloba was introduced in Europe in the 18th century, sometime around 1730. The tree surviving in the botanicalgarden of Utrecht is among the first ones that arrived in Europe. On September 27, 1815 Goethe wrote a poem inHeidelberg, which is attributed to the glory of this tree. On the basis of material contained in certain publications [11],proper prescription ofG. biloba leaves for internal medical use in China seems to have been first mentioned in the bookof Liu Wen-Tai in 1505 A.D [12]. The fruit (baigo) is in use since ages and remark on its leaf extract is included in themedical Dictionary of the Republic of China.

G. biloba is a member of the mixed mesophytic forest community that covered the hill country bordering theYangtze River Valley in China [13,14]. One of the last wild plants of G. biloba is reported to be in Zhejiang Province,China on the west peak of Tianmu Mountain (1506 m amsl). Ginkgo was introduced in Europe around 1730 and iswidely cultivated as an ornamental tree in streets and parks and as a medicinal plant particularly in China, Korea,France, Germany and the United States. The medicinal properties of Ginkgo seeds are reported in ‘Pen Ts'o Kang Mu’[15]. A recent survey by the authors has revealed that there are about 30 plants of G. biloba planted all over India,mostly in hilly tracts from north-west to north-east.

4. Ecology

The Tianmu Mountain in China the Ginkgo population is considered about 244 individuals with a mean diameter of45 m at breast height (DBH) and a mean height of 18.4 m. Most of the population of Ginkgo was growing on degradedsites, along stream beds, on rocky slopes and on the edges of soil beds. Ginkgo seedlings are quite rare on TianmuMountains and are found in areas of the forests that have been highly disturbed. In some areas, the exceptional speciesrichness of the surrounding forest and the large size of many of its trees suggest that they may be well developed [16,17].Recent studies on its population biology indicate a relatively low degree of genetic diversity among the plants. About40% of the TianmuGinkgos have more than one trunk with DBHmore than 10 cm.Most of these secondary trunks haveoriginated from lignotubers located at or just below ground level. Secondary trunk formation is most apparent inspecimens that were damaged due to severe stress. The sprouting ability of Ginkgo is an important property that hassupported the species to perpetuate on the eroded mountains and in survival and morphological stability of the species.

5. Sexual reproduction

Ginkgo is a dioecious species, with male and female plants occurring in about 1:1 ratio, with sporadic report ofmonoecious individual [18]. Its leaves are deciduous, alternate or in clusters of 3–5 on short twigs, petiolate, fan or

404 B. Singh et al. / Fitoterapia 79 (2008) 401–418

Adiantum shaped, bilobed, thickened at the margin, 5–10 cm, across, broader than long with venation dichotomouslybranching seemingly parallel.

The name of Ginkgo has been derived from a wrong transcription of the Japanese name Yin-Kwo, meaning Silverfruit. The name of the species biloba, recalls the bilobed shape of the leaves. The resemblance of the shape and vein ofits leaf with those of the leaf of maidenhair fern has established its common English name. In China and Japan, it isconsidered sacred and is cultivated around temples. The male trees are more desirable for planting, because femaleplants produce foul-smelling seeds. G. biloba is a tree of great beauty with a long life span, highly resistant to insects,bacterial and viral infections and air pollution. Ginkgo shows a long juvenile period, typically not reaching sexualmaturity until 20 to 30 years. Male and female sex organs are produced on short shoots, in the axils of bud scales andleaves. The male catkins emerge before the leaves and fall off immediately after shedding their pollen. Wind pollinationoccurs during early April in the regions with mild winters, and during late May in areas with severe winters. As thepollination ceases, it is difficult to say what is the maximum limit of dispersal of pollens, however in the Boston area nosetback to seed setting was recorded even when the male and female trees were 400 m apart [19]. Its ovules are 2 to3 mm long and produced in pairs at the end of stalks 1.5 to 2.0 cm long. In receptive stage, it secretes a somewhatmucilaginous fluid from its micropyle, which facilitates trapping airborne pollen chamber. Once inside the ovule, themale gametophyte commences a 4 month long development period that culminates into production of a pair ofmultiflagellated spermatozoids, one of which fertilizes a waiting egg cell [20] while the ovules are still on the tree [21].Depending upon the date of pollination, the union can occur anytime from late August to late September. Ginkgo seedat maturation is large and consists of an embryo encircled, in the tissue of the female gametophyte covered by a thickseed-coat layer. The seed-coat consists of a soft, fleshy outer layer (sarcotesta), a hard, stony middle layer and a thinmembranous inner layer. The seed contains fleshy sarcotesta, which is generally known as the Ginkgo nut. Thedeveloping ovules of Ginkgo are green in color up to maturation, when owing to cold temperature they attain yellowcolor as that of the leaves. The seeds drop from the tree about a month after fertilization. The foul odor from Ginkgoseeds comes out only after they are fully mature. The sarcotesta contains two volatile compounds viz., butanoic andhexanoic acids. The seeds also contain phenolic compounds causing contact dermatitis in humans [2].

6. Seed dispersal and establishment

It is a mystery about the animal which might have dispersed Ginkgo seeds during the course of its evolution. Severalworkers perceive that dinosaurs might have dispersed Ginkgo seeds, but none of them have provided any evidence tosupport the claim [2]. It is believed that the early mammals in the extinct family Mutituberculata could have beeneffective dispersal agents [19]. These marsupials like creatures, often referred to as the ‘rodent of the Mesozoic’, werefound in the temperate parts of the northern hemisphere from the late Jurassic through the Oligocene. At present, somany mammals are observed feeding on, and presumably dispersing, the odoriferous, nutrient-rich seeds of G. biloba.In Rodentia order, these include the red-bellied squirrel (Callosciurus flavimanus var. ning poemsis, family Sciuridae)in the region of Tianmu Mountain [16], and gray squirrel (Sciurus carolinensis, family Sciuridae) in eastern NorthAmerica [19]. In Carnivora order, potential dispersal agents assumed the masked palm civet (Paguma larvata, familyViveridae) in the Tianmu Mountain area. The leopard cat (Felis bengalensis, family Felidae) in Hubei Province, China[19] and the raccoon dog (Nyctereutes procyonoides, family Conidae) in Japan [22].

The existence of three independent reports of carnivores consuming whole Ginkgo seeds and defecating intact nutsraises the possibility that the foul foul-smelling sarcotesta may be attracting these animals [16]. Ginkgo seeds aredormant when they fall from the tree because the embryo is not fully developed, and are only 4 to 5 mm in length. Incase seeds are collected shortly after dispersal, cleaned, and placed in a warm greenhouse, the embryo will grow to itsfull size i.e. 10 to 12 mm in length and germinate within 8 to 10 weeks [21] In the warm-temperate climate, Ginkgoseeds are shed in late summer, whereas under cold temperature seeds are shed late in the season. The colder temperaturedelays full embryo development until the ensuing spring.

7. Tree architecture

G. biloba is admitted as a long lived deciduous tree. Its stem diameter has been recorded to range from 1 to 4 m [23],depending upon the agro-climatic conditions. A mature tree may attain up to 20 to 40 m height. However, oneexceptional specimen in Korea is reported to be 64 m tall with 14 m diameter [24]. The appearance of a fully developed

405B. Singh et al. / Fitoterapia 79 (2008) 401–418

young Ginkgo tree is distinctly pyramidal with a dominant central trunk and widely spaced diagonal orientatedbranches in whorls. The sexual maturity takes about 25 years. Vertical growth generally slows down and the tree fills inits sparsely branched Juvenile structure, with a broad, spreading canopy [25,26].

After about 10 years of growth, Ginkgo produces two types of shoots; long shoots with widely spaced leaves thatsubtend axillary buds, and short shoots with clustered leaves that lack both internodes and axillary buds. The longshoots are basically responsible for over all growth of the tree and generating new growing points. The short shootsproduce clusters of leaves with reproductive structures. In due course of time, the effect of environmental andphysiological factors on the growth pattern of these shoots can be changed i.e. short shoot can produce a long shoot,and the terminal growth of a long shoot may be restricted to produce a short shoot [25]. This alternation provides asimple mechanism for modulating carbohydrate distribution between sexual reproduction and vegetative growth [26].

Under adverse growing conditions, both in stress and in higher accumulation of food material, Ginkgo is capable ofproducing secondary trunks, may be 2–3 in number, just below ground level. Generally, these secondary stemsoriginate from root like positively geotropic shoots known as lignotubers or ‘basal chichi’ or by simple buds just belowthe soil surface. Anatomically lignotubers are developed in all Ginkgo seedlings as part of their normal ontogeny frombuds located in the axils of the two cotyledons, and also with vegetative propagated plants by using stem cuttings.

8. Seed production

The seed of G. biloba contains a thin-layer nut, which is consumed as food and a medicine throughout the world.Fresh nuts contain about 41% moisture. Dry nuts contain 6% sucrose, 68% starch, 13% protein and 3% fat [27]. Thecommercial production of Ginkgo nuts has been reported in China for more than 600 years. About 44 cultivars withbest performance in growth have been selected, based on quality, size and shape of their nuts and productivity [2,28].These cultivars are propagated by grafting technique using seedling rootstocks. These grafted plants start bearing nutsafter 5 years of age. In China, a widely grown cultivar, Dafushon, in Jiangsu Province, produces 5 to 10 kg of nuts by a15 year old tree and between 50 to 100 kg by a 50 year old tree. One recent production from 700,000 to 800,000 treesproduced an average of 6000–7000 tons of dry nuts per year [2].

9. Leaf production

Recently (1982), G. biloba has been planted on a large scale in France (480 ha) and the United States (460 ha). Forthis purpose, the saplings are planted at a closer spacing of 40 cm in rows at 1 m distance accommodating 25,000 plantsper hectare. Height of the plantation is maintained at about 3 m by deploying special pruning technique to facilitatemechanical harvesting of leaves. Green leaves were harvested in summer season. In a rotation of 4 to 5 years of cropseason, the plants are pruned to near ground level, after harvesting. After such pruning or cutback operation, newshoots sprout from buds located at the base of the stem or on basal lignotubers. With proper irrigation and fertilizersapplication in growing season, the annual vertical growth from the point of cutting is generally 1 m or even more [25].In Eastern China, after 1992, Ginkgo seedlings have been established about 2000 ha area specifically for commercialleaf production. These plantations began producing marketable quantities of leaves in 1996, and the estimated yieldwill be increased dramatically over the next 5 to 10 years [2,29].

10. Cultivation and propagation

Perennial plantations are raised with a plant to plant spacing of 3 to 6 m. It prefers full sunlight and well drained soilwith mean soil temperature 15 to 27 °C. Ginkgo exhibits resistance to infection and air pollution. Fifty million G.biloba trees are grown especially in China, France and South Carolina, USA, producing over 8000 tons of dried leaveseach year to meet the commercial demand.

A tree of Ginkgo is known to be over 5000 years old [30]. A study on effect of physical treatment on germination ofG. biloba seeds has confirmed that the seed viability decreased over time [31]. There are studies on effect of foliarapplication of nitrogen on 3 year old G. biloba seedlings [32] and on effect of nitrogen, phosphorus and potassium onleaf and vegetative growth of G. biloba [33,34]. Fang Ren et al. [35] have examined the microclimatic observationsand intercropping system with G. biloba. Youchao et al. [36] have studied the coppice management to improve theyield and quality of G. biloba leaf producing plantation. Quan BingYan et al. [37], Wang et al. [38] and Kang Zhi et al.

Table 1Secondary metabolites reported from G. biloba root bark, wood, and leaves

Material Reference

TerpenesMonoterpenes [64]Cymene (1)Isopropyl-phenol (2)Thymol (3)Linalool oxide (4)Ionone (5)

Diterpenes [68,69]Ginkgolide A (6)Ginkgolide B (7)Ginkgolide C (8)Ginkgolide J (9)Ginkgolide M (10)Ginkgolide K (11)Ginkgolide L (12)

Sesquiterpenes [64,69]Bilobalide (13)Bilobanone (14)E- and Z- forms of 10-11-Dihydroatlantone (15)E-10-11-Dihydro-6-oxoatlantone (16)Elemol (17)Eudesmol (18)

Steroids, phytosterols [64]β-Sitosterol (19)Stigmasterol (20)Campesterol (21)Dihydrobrassicasterol [64]

Carotenoids [64]α-Carotene (22)γ-Carotene (23)Lutein (24)Zeaxanthin (25)

Polyprenols [64,69]Ginkgo polyprenols (26)Ginkgo polyprenol acetates (27)

FlavonoidsGlycosides of [64,69]Kaempferol (28)Quercetin (29)Myricetin (30)Apigenin (31)Isorhamnetin (32)Luteolin (33)

AglyconesKaempferol(28)Quercetin (29)Myricetin (30)Apigenin (31)Isorhamnetin (32)Luteolin (33)Tamarixetin (34)4′-OMe Apigenin (35)3′-Methylmyricetin (36)Catechin (37)Epicatechin (38)Epigallocatechin (39)Gallocatechin (40)

406 B. Singh et al. / Fitoterapia 79 (2008) 401–418

Table 1 (continued )

Material Reference

Dimers [64]Catechin–catechinEpicatechin–catechinEpigallocatechin–catechinGallocatechin–catechin

Anthocyanidin [69]Procyanidin (41)Prodelphinidin (42)

Biflavones [64,69]Amentoflavone (43)7-Methoxyamentoflavone (44)Bilobetin (45)5′-Methoxybilobetin (46)Sequojaflavone (47)Ginkgetin (48)Isoginkgetin (49)Sciadopitysin (50)

Biflavone glucosides [70]GinkgetinIsoginkgetin

Alkyl phenols and alkyl phenolic acids [64]Cardanols3-Tridecylphenol (51)3-Tetradecylphenol (52)3-Pentadecylphenol (53)3-Heptadecylphenol (54)Ginkgol (55)

Cardols [64]5-Tridecylresorcinol (56)5-Tetradecylresorcinol (57)5-Pentadecylresorcinol (58)5-Heptadecylresorcinol (59)Bilobolol (60)

Anacardic acids [64]6-Tridecylsalicyclic acid (61)6-Tetradecylsalicylic acid (62)6-Pentadecylsalicylic acid (63)6-[8-Pentadecenyl] salicylic acid (64)6-Hexadecylsalicylic acid (65)6-[9, 12-Heptadecadienyl] salicylic acid (66)6-[8-Heptadecenyl] salicylic acid (67)

Resorcylic acids [64,69]6-[8-Pentadecenyl] resorcylic acid (68)6-Tridecylresorcylic acid (69)Alkyl coumarin (70) [64,69]

Organic acidsAscorbic acids (71)D-glucaric acid (72)Quinic acid (73)Shikimic acid (74)6-Hydroxykynurenic acid (6-HKA) (75)p-Hydroxybenzoic acid (76)Protocatechuic acid (77)Vanillic acid (78)Acetic acidButyric acidFormic acidHexanoic acid

(continued on next page)

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Table 1 (continued )

Material Reference

Organic acidsValeric acid

Carbohydrates [64]SucrosePinitol (79)Sequoyitol (80)Polysaccharides

Long chain hydrocarbons and lipids [64]1-Hexacosanol2-Hexenaln-Nonacosane (81)n-Nonacosan-10-ol (ginnol) (82)n-Nonacosan-10-one (ginnon) (83)n-Octacosan-1-olLinolenic acidLinoleic acids14-Methylhexadecanoic acid5, 9, 12-Octadecatrienoic acidOleic acidPalmitic acidEicosanedicarboxylic acidPhellonic acid

Inorganic salts/complexes [64]SeleniumCopperZincChromiumIronManganeseCalcium oxalatePhosphoric acid

Miscellaneous organic compounds [64]N-containing compoundsAminobutyric acidAsparagineAspartic acidGlutamineArginineProlineGlycine4-O-Methylpyridoxine (84)ProlinePyroglutamic acidSerineThreonineValineIsopentenyladenineRibosylzeatinZeatin and dihydrozeatin (85)Zeatin glucosideHexosamines6-Hydroxykynurenic acidLectinsPentadien-1, 5-diyl-diphenol

Others [64]TanninsCondensed tanninsGlycerol derivatives1-Hydroxypyrene conjugates

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[39] have studied the effect of density and stump height on leaf yield of G. biloba. Huang et al. [40] and Sung ChiungHuei et al. [41] have evaluated it for absorption and tolerance to ozone and nitrogen dioxide. Li Jian et al. [42] andLeng-PS et al. [43] have studied the effect of intensity and quality of light on photosynthesis of G. biloba.

Ginkgo tree is an extremely slow growing one and its regeneration through seeds is very poor. Therefore, vegetativemultiplication through cuttings is the only possible option for augmenting regeneration. The stimulation of adventitiousroot formation in stem cuttings treated with auxins and commercial rooting mixture containing auxin is well known[44]. Li et al. [45] have obtained best result when G. biloba cuttings were treated with carbendazim. However,crystalline potassium permanganate and IAA (Indole acetic acid) containing powder have also been recommended forstimulating rooting of G. biloba [46]. In China, dipping the base of cutting into ABT-1 (aminobenzotriazole) rootingpowder for 1 h gave the best results [47,48]. In addition to auxins, phenolic compounds either alone or in combinationof auxins [49,50] have also stimulated adventitious root formation in cuttings of several species. Shen Xiao Qing et al.[51] have studied the propagation by shoot cutting of G. biloba in clay pots. Om Parkash et al. [52] have tried IBA(Indole-3-butyric acid) along with certain phenolics for root induction in stem cutting of G. biloba. Schmid [53] hasstudied cultivation of G. biloba. Yu Xin et al. [54] have studied storage technology of G. biloba seeds at normalatmospheric temperature. Wu [55] have has studied the techniques for growing seedlings of G. biloba. Lee and Lee[56] have reported the optimal planting spacing on the basis of growth condition of landscape trees.

11. Pharmacological importance

Although Ginkgo tree has been around for 200 million years, it's only during the last couple of decades that its truevalue has been recognized. Its amazing vitality has attracted an increasing exploration into potential application inhealth, foods and supplements. The medicinal parts of Ginkgo tree are fresh or dried leaves and seeds separated fromtheir fleshy outer layer. It contains large number of active compounds, the most important of which are flavanolglycosides and terpene trilactones. The extract of Ginkgo leaves are standardized for these two components. Activecompounds in Ginkgo extract improve blood circulation, discourage clot formation, reinforce the walls of thecapillaries, and protect nerve cells from harm when deprived of oxygen. The leaves extracts are used to treat dementialdisorder, such as concentration difficulties and memory impairment. The extract also possesses antioxidant [57],antiasthmatic [58], scavenge radicals [59], wound healing [60] and neuroprotective properties as well as it improvesmental capacities in Alzheimer's patients [61,62].

12. Economical importance of the plant

Western medical interest in G. biloba has grown dramatically since the 1980s owing to its potent action oncardiovascular system of human beings, particularly on the cerebral vascular activity. Over 7 billion dollars are spentannually on botanical medicines and Ginkgo ranks first among herbal medications [57]. Fifty million G. biloba treesare grown, especially in China, France, and South Carolina, USA, producing 8000 tons of dried leaves each year to

Fig. 2. Structures of secondary metabolites of G. biloba.

Fig. 2 (continued ).

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meet the commercial demand for G. biloba products [57]. Ginkgo is among the most sold medicinal plants and theannual consumption in 2001 was between 4.5 million pounds and 5.1 million pounds of dried leaves [63]. The use ofG. biloba has been growing at a very rapid rate world wide at 25% per year in the open world commercial market.Germany, Switzerland and France have respectively 31%, 8% and 5% of the world commercial market. Presently thereare around 142 G. biloba products on the global market and it is estimated that in the coming five years its utilization isexpected to grow threefold. G. biloba is sold in the form of leaf, powder extract, and as a tincture to the pharmaceutical

Fig. 2 (continued ).

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Fig. 2 (continued ).

412 B. Singh et al. / Fitoterapia 79 (2008) 401–418

and herbal companies [64]. Here in this report we have attempted to compile the information available on extractionprotocols and on the estimation of terpene trilactones and flavonoids in G. biloba.

13. Secondary metabolites

G. biloba has been well investigated chemically for various classes of constituents. It is reported to contain a numberof secondary metabolites including terpenoids, polyphenols, allyl phenols, organic acids, carbohydrates, fatty acids andlipids, inorganic salts and amino acids. However terpene trilactones and flavonoids are considered the main bioactiveconstituents. Composition and analytical procedures have also been well documented in different pharmacopoeias[65–67]. These metabolites are listed in Table 1.

Ginkgolides, a terpene trilactones, are compounds with cage like structure. Furukawa (1932) isolated for the firsttime ginkgolide A, B, C and M fromG. biloba root bark. The ginkgolide A, B, C and J are also reported from the leavesofG. biloba [64]. It is interesting to note that it was found only in leaves whereas ginkgolide M is found only in the root

Fig. 2 (continued ).

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bark of G. biloba [57]. In addition to the ginkgolides (diterpenes), a bilobalide (sesquiterpene) is also found in G.biloba. Bilobalide is closely related to the ginkgolides [64]. Structures of ginkgolides K and L were establishedrecently. Ginkgolides and bilobalide are collectively known as terpene trilactones (TTLs). Terpene trilactones arereported to be present only in G. biloba. To the best of the knowledge until now terpene trilactones are the only naturalproducts possessing t-butyl group in their structure [71]. Therefore, terpene trilactones has received more attentionamong other constituents of G. biloba. Ginkgolides are the diterpenes having cage like structure consisting of six 5-membered carbocyclic rings, three lactones and a tetrahydrofuran. Bilobalide is a sesquiterpene and differs by theabsence of tetrahydrofuran ring [64] (Fig. 2).

Fig. 2 (continued ).

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Terpene trilactones are the cage like compounds and are extraordinarily stable despite the presence of the multipleoxygen functionals. They are unaffected by boiling in HNO3 [57]. The crude leaf extract contains significant amount ofmore apolar compounds like ginkgolic acid, biflavones and chlorophyll along with active ingredients. Various methodsof extraction have been published for the enrichment of bioactive constituents and are listed in Table 2.

The quantities of terpene trilactones can vary greatly with small changes in parameters such as collection site,harvest time and plant growth stage [64,76,82]. Analysis of terpene trilactones, including bilobalide and ginkgolides A,

Table 2Extraction and sample enrichment methods of terpene trilactones

Methods References

H2O2 followed by extraction with EtOAc. [72]0.1% Na2HPO4, pH = 8 [73]Pressurized water extraction [74]Supercritical CO2 [75]Polyamide, C18 SPE column [72,76]Liquid–liquid Extraction with EtOAc–THF [77]Amberlite XAD-7HP intermediate polar resin [78]Supercritical fluid extraction [79]Glass fiber membrane modified with [80]OctadecyltrichlorosilaneNanofiltration [81]

H2O2, hydrogen peroxide; EtOAc, Ethylacetate; Na2HPO4, di-sodium hydrogen phosphate; CO2, carbon dioxide; SPE, solid phase extraction; THF,tetrahydrofuran.

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B, C and J has long been difficult because of poor UV absorption due to the lack of good chromophore and relativelylow concentrations in leaves. For the analysis of these compounds, various quantitative methods based on HPLC, GC,LC–MS, capillary electrophoresis, TLC have been published and details are given here in Table 3.

Table 3Methods of analysis of terpene trilactones

Methods Solvent system References

Thin-layer chromatography Toluene:acetone (7:3) [69]C6H6:EtOAc (1:1) [69]EtOAc:toluene:acetone:hexane (4:3:2:1) [69]CHCl3:acetone:HCO2H (75:16.5:8.5) [69]Toluene:EtOAc:acetone:MeOH (10:5:5:0.6) [69]EtOAc:MeOAc (1:1) [65]

Liquid chromatography RP–HPLC–UV MeOH:iPrOH:H2O (15:10:75) (preparative) [83]MeOH:ACN:0.01 M phosphate buffer (pH=5.0) [84]H2O:MeOH:iPrOH (72.5:17.5:10) [85]H2O:CAN:THF (10:2:1) RP–HPLC–RI [78]H2O:MeOH:THF (7:2:1) [72]MeOH:H2O:H3PO4 (25:75:0.1) [86]MeOH:H2O (33:67) RP–HPLC–ELSD [87]NH4OAc:MeOH:iBuOH [88]MeOH:H2O (gradient) [82]

HPLC–ELSD EtOAc:Pet. ether:MeOH (55:40:5) [89]LC–ESI–MS THF:iPrOH:H2O (4:8:88) (+ve ion mode) [90]LC–MS–MS H2O:MeOH (−ve ion mode) [91]LC–APCI–MS H2O:MeOH (−ve ion mode) [92, 93]LC–SSI–MS H2O:MeOH [94]

By derivatization benzylation–debenzylation [95]Gas chromatography GC-FID Conditions: [15,96]

Column DB-5 Injector temp — 280 °CDetector temp — 300 °COven temp — 200 °C 0–1 min280 °C 1–5 min

Super critical fluid chromatography CO2 modified with 10%MeOH [97]H2O:THF:MeOH (68.5:10.5:21)Oven temperature — 40 °CPressure — 280 atm

Capillary electrophoresis Capillary — 60 cm×75μm, 30 °C [76] [76]Buffer 25 mM phosphate and 90 mM SDS

NMR Acetone d6:benzene d6 (50:50) [98]

C6H6, cyclohexane; CHCl3, chloroform; HCO2H, formic acid; MeOH, methanol; MeOAc, methyl acetate; iPrOH, isopropyl alcohol; H2O, water;ACN, acetonitrile; H3PO4, phosphoric acid; iBuOH, isobutyl alcohol; NH4OAc, ammonium acetate.

Table 4List of extraction and analytical methods of flavonoids

Method References

Molecularly imprinted polymer (MIP) [99]Leaching with aqueous two phase system [100]Adsorption separation [101]Ultrafiltration [102]RP–HPLC–UV [69,82,66,67]CE–ED [103]HPTLC [104,105]HPLC–PDA [106]LC–MS–MS [107]

RP–HPLC–UV, Reverse phase–high performance liquid chromatography–ultra violet spectrometer; CE–ED, capillary electrophoresis–electrochemical detection; HPTLC, high performance thin thin-layer chromatography, PDA, photo diode array; LC–MS, liquid chromatography–mass spectrometry.

Table 5List of methods of simultaneous analysis of terpene trilactones and flavonoids

Method References

RP–HPLC–ELSD [108]RP–HPLC (inline UV–ELSD) [109]GC–MS [110]Reverse-flow micellar electrokinetic [111]Chromatography (RF–MEKC)Nano ESI–MS [112]NMR [113]

ELSD, evaporative light scattering detector; GC, gas chromatography; ESI, electrospray ionizations; NMR, nuclear magnetic resonance.

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13.1. Flavonoid glycosides

Flavonoids are the polyphenolic, low molecular weight compounds, probably found in all green plants. Currently,more than 30 genuine flavonoids are known in G. biloba. The great number of different flavonoids is not a result of thevariability of the 2-phenylchromane framework but of the different glycosides found in Ginkgo. Nevertheless, onlyglucose and rhamnose can be found as sugar molecules and the variety of mono-, di- and triglycosides in differentbinding patterns [63]. The flavonoids comprise of large group of polyphenols and include flavones, flavonol glycosides,acylated flavonol glycosides, biflavonoids, flavan-3-ols and proanthocyanidins. Of these flavonol glycosides present inG. biloba leaves are more abundant than the other flavonoids and most of them being derivatives of quercetin,kaempferol and isorhamnetin. The aglycones themselves occur only in relatively low concentration (Table 4).

A good number of publications are available on quantitative analysis of ginkgoterpenes and flavonoids individuallywhich is time consuming and uneconomical. Few reports which have appeared on simultaneous quantification arelisted below in Table 5.

Acknowledgements

Authors are grateful to the Director IHBT, for providing necessary facilities. Financial assistance to PushpinderKaur from the National Medicinal Plant Board, DBT, Government of India, is gratefully acknowledged.

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