Hydrolyzable tannins of tamaricaceous plants. IV: Micropropagation and ellagitannin production in shoot cultures of Tamarix tetrandra

Phytochemistry 72 (2011) 1978–1989

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Hydrolyzable tannins of tamaricaceous plants. IV: Micropropagation and ellagitannin production in shoot cultures of Tamarix tetrandra Mohamed A.A. Orabi a,c, Shoko Taniguchi a, Susumu Terabayashi b, Tsutomu Hatano a,⇑ a

Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima, Okayama 700-8530, Japan Laboratory of Medicinal Resources, Department of Kampo Pharmacy, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka, Yokohama, Kanagawa 245-0066, Japan c Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt b

a r t i c l e

i n f o

Article history: Received 13 January 2011 Received in revised form 9 July 2011 Available online 8 August 2011 Keywords: Tamarix tetrandra Tamaricaceae Shoot culture Micropropagation Polyphenol Ellagitannin Ellagitannin biosynthesis Plant laccases

a b s t r a c t Shoot cultures of Tamarix tetrandra on Linsmaier–Skoog (LS) agar medium with 30 g l 1 sucrose, 2.13 mg l 1 indoleacetic acid and 2.25 mg l 1 benzyl adenine produced ellagitannins found in intact plants of the Tamaricaceae. This was demonstrated by the isolation of 14 monomeric–tetrameric ellagitannins from the aq. Me2CO extract of the cultured tissues. This is the first report on the production of ellagitannin tetramers by plant tissue culture. The effects of light and certain medium constituents on tissue growth and ellagitannin production were examined. The contents of representative tannins of different types [i.e., tellimagrandin II (monomer), hirtellin A (linear GOG-type dimer), hirtellin B (hellinoyltype dimer), hirtellin C (macrocyclic-type dimer), and hirtellin T1 (linear GOG-type trimer)] in the resultant tissues in response to these factors were estimated by HPLC, and the optimal condition for production of these tannins were established. Shoots cultured on LS hormone-free medium promoted root development, and regenerated plants could adapt to ordinary soil and climate. Acclimatized and intact T. tetrandra plants that were collected in November and May, respectively, demonstrated seasonal differences in individual ellagitannin contents. HPLC comparison of individual ellagitannin contents in different plant materials (i.e., leaves, stems, and roots) of intact T. tetrandra plants is also reported. The results are discussed with respect to cellular deposition and biosynthetic relationship of tannins. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Plant species of the genus Tamarix L. (Family: Tamaricaceae) are commonly known as tamarisks or saltcedars. Tamarix generally occurs in dry, saline habitats in subtropical and temperate zones from western Europe and the Mediterranean to North Africa, northeastern China, India, and Japan, and several species have also been found in the United States. Tamarix is cultivated as ornamental plants in gardens for its pleasing racemes of small pink to whitish flowers. Various medicinal uses and biological activities of Tamarix species have been described in a review article (Sharma and Parmar, 1998) and in several papers (Sultanova et al., 2001, 2004; Saïdana et al., 2008; Abouzid et al., 2009). Phytochemical studies, including earlier studies on Reaumuria hirtella and Tamarix pakistanica (Yoshida et al., 1991a,b, 1993a,b; Ahmed et al., 1994a,b) and our recent investigations of Tamarix nilotica (Orabi et al., 2009, 2010a,b) have demonstrated the occurrence of a wide class of hydrolyzable tannins in tamaricaceous plants. Among the tannins isolated from these species, hirtellins A (1) and B (2), and tamarixinin A (3) exhibit significant host-mediated ⇑ Corresponding author. Tel.: +81 86 251 7936; fax: +81 86 251 7926. E-mail address: [email protected] (T. Hatano). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.07.011

antitumor activity against sarcoma 180 in mice (Miyamoto et al., 1993). Hirtellin A (1) and nilotinin D8 show prominent selective cytotoxic effects against a broad spectrum of human tumor cell lines, including human squamous cell carcinomas (HSC-2, HSC-3, and HSC-4), and human promyelocytic leukemia (HL-60) cells (Orabi et al., 2010b). Stimulation of peripheral blood monocyte iodination has been reported for hirtellin E and remurins A (4) and B (Sakagami et al., 1992). Tamarix tetrandra Pall. ex M. Bieb. (Gaskin and Schaal, 2003; Fakir, 2006; Tashev and Taskov, 2008; Hamzaog˘lu and Aksoy, 2009), commonly known as small-flowered tamarisk, is widespread in southern Europe, United States, and Asia Minor. It has been used as a windbreak along the seacoast and also for arresting soil erosion. Despite the lack of reports on tannin constituents of T. tetrandra, HPLC comparisons of aq. Me2CO extracts of leaves from T. tetrandra and T. nilotica show similar ellagitannin profiles. To further assess biological activities of tamaricaceous ellagitannins, finding an alternative source for the stable production of these tannins through a biotechnological approach has become increasingly important. In this context, we established shoot cultures of T. tetrandra on Linsmaier–Skoog (LS) (Linsmaier and Skoog, 1965) agar medium capable of producing ellagitannins. Chromatographic separations of an aq. Me2CO extract of the cultured shoots produced

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

14 monomeric–tetrameric ellagitannins belonging to different tamaricaceous tannins. In this study, we examined tissue growth, ellagitannin constituents, and influence of culture conditions and manipulating nutri-

1979

ents in the culture medium on the growth and tannin production of T. tetrandra cultured shoots. An HPLC comparison of individual ellagitannin contents in the aq. Me2CO extracts of leaves from intact T. tetrandra plants, which were collected during autumn and

Fig. 1. Structures of ellagitannins (1–14) isolated from cultured shoots of T. tetrandra.

1980

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

early summer, was attempted. We also estimated the individual ellagitannin contents of leaves, stems, and roots from intact T. tetrandra plants by HPLC. This paper summarizes these results and the production of tamaricaceous ellagitannins using cultured T. tetrandra shoots. Although micropropagation of Tamarix gallica from nodal explants has been reported (Lucchesini et al., 1993), this is the first study on tannin production of a tamaricaceous plant using in vitro culture.

2.2. Ellagitannins of cultured shoots An aq. Me2CO extract of fresh TtD strain tissues, which were harvested 30 days from the inoculum time, was subjected to chromatography on Diaion HP-20, Toyopearl HW-40 (coarse grade), Sephadex LH-20, and MCI-gel CHP-20P gels, this being followed

(a) Content (mg/g fr. wt)

2.1. Establishment of shoot cultures of T. tetrandra

(2.3)

(2)

(1.8)

(2)

1 0.8 0.6

2 11 13

0.4 0.2 0 10

Content (mg/g fr. wt)

T. tetrandra callus and shoot cultures were successfully induced from segments of young actively growing stems of a cultivated T. tetrandra tree. Stem-explants were cultured on LS medium supplemented with 30 g l 1 sucrose and 10 g l 1 agar added with six auxin/cytokinin combinations to produce calli and shoots. The LS medium with combinations of indoleacetic acid (IAA)/kinetin (KIN), and IAA/benzyl adenine (BA) induced shoot development. The best results for shoot production were obtained with the latter combination; shoots grew ca. six times (2.3 g, average of 6 replicates from two trials) the inoculum weight (0.35 g) in 30 days under light illumination. The other auxin/cytokinin combinations (see Section 5.2) alternatively promoted calli development, but showed slower proliferation than shoots grown under the above conditions. The ellagitannin profiles were checked by extraction and subsequent HPLC analyses that compared authentic samples, which were isolated from T. nilotica in our preceding study (Orabi et al., 2009, 2010a,b) and were practically identical for the different callus and shoot lines. Shoots induced on a medium with an IAA/BA combination were maintained by routine subculture at 30-day intervals on fresh LS agar media with the same sugar and hormone components for more than 3 years. Cultures were incubated at 25 °C under light illumination to produce the T. tetrandra light (TtL) strain and in the dark to produce the T. tetrandra dark (TtD) strain (see Section 5.2). The growth and tannin content of the tissues remained unchanged during this period.

1.2

8

6 1

2

6 4

0

TtL-L

(b)

3.5

Content (mg/g fr. wt)

2. Results

(3.9)

TtL-D

(1.8)

TtD-D

(3.7)

TtD-L

(1.8)

3 2.5 2 1.5

2 1 11 0.5 13 0

0.5 (0.9)

(1.4)

(2.0)

(2.3)

(2.6)

(2.7)

2 11 13

Content (mg/g fr. wt

Content (mg/g fr. wt)

14

0.4 0.3 0.2

0

Content (mg/g fr. wt)

4

8

4 2 0

12

6 1

10

6

6 1

0.1

10

12

TtL-S

8 6 2 0 7

14

21 28 Time (day)

35

42

Fig. 2. Time course of tissue growth and individual ellagitannin (1, 2, 6, 11, and 13) content of cultured tissues of T. tetrandra. Tissue growth (g fr. wt/test tube) is presented by the numbers shown in parentheses at the top of the graph. The bars indicate the s.e. of the means of three replicates. Tannins 2, 11, and 13 are produced in low amounts by the cultured tissues and are shown in the upper part of the graph, and tannins 1 and 6 are produced in larger amounts and are shown in the lower part of the graph.

TtL-U

TtD-S

TtD-U

Fig. 3. Effects of light on tissue growth and ellagitannin (1, 2, 6, 11, and 13) contents of cultured tissues of T. tetrandra. Tissue growth (g fr. wt/test tube) is presented by the numbers shown in parentheses at the top of the graph. The bars indicate the s.e. of the means of three replicates. (a) Shoots from the T. tetrandra light (TtL) grown tissues were cultured on LS agar medium containing 30 g l 1 sucrose, 2.13 mg l 1 IAA and 2.25 mg l 1 BA, and were grown under light illumination (12 h/day, fluorescent lamp 3000 l) and dark conditions to produce TtL-L and TtL-D, respectively. Similarly, shoots from the T. tetrandra dark (TtD) grown tissues were cultured on LS agar medium, with the same sugar and hormone component, in dark and under light illumination (12 h/day, fluorescent lamp 3000 l) to produce the TtD-D and TtD-L, respectively. (b) Shoots (1 g) from the TtL and TtD tissues were cultured on 30 ml LS agar medium containing 30 g l 1 sucrose, 2.13 mg l 1 IAA and 2.25 mg l 1 BA in a 100-ml flask, and were grown at 25 °C under light illumination (12 h/day, fluorescent lamp 3000 l) and dark conditions, respectively. The resultant cultures of the TtL strain consisted of shoots (TtL-S) and undifferentiated (TtL-U) parts. Similarly, cultures of the TtD strain consisted of shoots (TtD-S) and undifferentiated (TtD-U) parts.

1981

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

by further preparative HPLC purification to produce 14 ellagitannins. As shown in Fig. 1, these ellagitannins can be classified structurally into simple ellagitannin monomers (A), tellimagrandins I (5) (Orabi et al., 2010b) and II (6) (Orabi et al., 2010b), and several types (B–G) of tamaricaceous ellagitannins. Type-B includes ellagitannin monomers with a m-GOG moiety, 4 (Orabi et al., 2009), and nilotinin M2 (7) (Orabi et al., 2010a). Type-C includes linear GOGtype dimers in which the linking unit between the sugar cores is a m-GOG moiety, 1 (Orabi et al., 2009) and nilotinin D3 (8) (Orabi et al., 2009). Type-D includes a linear GOG-type dimer in which the linking unit between the sugar cores is a p-GOG moiety, tamarixinin C (9) (Yoshida et al., 1993b). Ellagitannins of type-E feature bridging of a hellinoyl (m-GO-m-GOG) moiety between the glucose cores, 2 (Orabi et al., 2010b) and 3 (Orabi et al., 2010b). Type-F includes macrocyclic dimers in which a macrocyclic ring including the glucose core carbons can be produced by bridging of two p-GOG moieties, tamarixinin B (10) (Yoshida et al., 1993b), m-GOG and p-GOG moieties, hirtellin C (11) (Orabi et al., 2010b), or two m-GOG moieties, isohirtellin C (12) (Orabi et al., 2010b). Type-G includes linear m-GOG-type oligomers, hirtellin T1 (13, trimer) (Ahmed et al., 1994a) and hirtellin Q1 (14, tetramer) (Ahmed et al., 1994a). Tannins 1–14 were identified by comparing normal- and reversed-phase HPLC elution profiles with authentic samples isolated from T. nilotica and/or comparison of NMR data with those in the literature. Furthermore, tannins with molecular weights corresponding to trimeric–pentameric tannins were estimated from their retention times (tR 13–32 min) on normal-phase HPLC (Okuda et al., 1989; Li et al., 2007) on comparison to those of 13 (tR 13 min) and 14 (tR 22 min) which were also detected in smaller amounts (see Section 5.3). Although the interglucose linkages in the isolated oligomers 13 and 14 were of the m-GOG-type, the presence of the p-GOG and hellinoyl moieties as linking units in the trimeric–pentameric tannins present in low abundance is also plausible, as their dimeric precursors were isolated in appreciable amounts (see Section 5.3). The results of the isolation process (see Section 5.3) and HPLC analyses of individual tannins shown below indicated that T. tetrandra tissues, which were cultured on ordinary LS medium, accumulated large amounts of the simple ellagitannins 5 and 6 and the linear m-GOG-type tannins 1, 8, 13, and 14, but low amounts of the hellinoyl-type tannins 2 and 3, and the tannins possessing p-GOG unit(s) in linear 9 or in macrocyclic modes 10 and 11. To identify optimal conditions for formation of these types of tannins, several factors effective for tannin production were examined, and the contents of representative tannins (1, 2, 6, 11, and 13) in the resultant cultures in response to these factors were estimated by HPLC as shown below.

thetic enzyme(s) and/or a gradual increase in the proportion of cells depositing 2 in the cultured tissues with time (see Section 3). 2.4. Effects of light on shoot growth and ellagitannin production The effects of light are shown in Fig. 3a. Although the weight of the tissues grown in the dark (TtD-D) was slightly lower than that of tissues receiving illumination (TtL-L), tannin accumulation was higher in tissues grown in the dark. Shoots transferred from light to dark (TtL-D) and the reverse (TtD-L) were intermediate in tissue weight and tannin production between those continuously grown under light and dark conditions. Because the obtained cultures were composed of undifferentiated stiff tissues and multiple shoots (see Section 5.2), they were separated from each other, and the tannin content in each part was analyzed by HPLC. As shown in Fig. 3b, the monomeric tannin 6 was most abundant in shoots of cultures grown under light illumination (TtL-S), whereas oligomeric tannins 1, 2, 11, and 13 were mostly localized in the undifferentiated parts of the cultures grown under light (TtL-U) and dark (TtD-U). Based on the results from the two experiments shown in Fig. 3a and b, the accumulation of 6 was enhanced by light, whereas oligomerization of tannins preferably occurred in the dark. Undifferentiated parts of the tissues accumulated large amount of ellagitannins. Ellagitannin production and cell growth of Heterocentron roseum callus cultures are known to be stimulated by light irradiation (Yazaki and Okuda, 1990), and light illumination also enhances production of galloylglucoses and the monomeric ellagitannins 5 and 6 in cultured Oenthera tetraptera shoots (Taniguchi et al., 2002b) and galloylglucose production in Liquidambar styraciflua callus cultures (Ishimaru et al., 1992). 2.5. Effects of manipulating nutrients in the culture medium Studies of effects of different nutrients on ellagitannin production were conducted on tissues cultured under light, as the content of 6, the primary ellagitannin metabolite in the biosynthesis of higher molecular weight ellagitannins, was high in the shoots of the TtL strain as described above. 2.5.1. Effects of Cu2+ The level of Cu2+ in the culture medium plays an important role in polyphenol production. This was demonstrated previously by the increased production of the C-glycosidic ellagitannins castalagin (15) and vescalagin (16) (Fig. 4) in a Quercus alba callus culture HO

OH

HO

OH

HO

OH CO

2.3. Time course of tissue growth and ellagitannin content

OC

O

The time course of tannin production and tissue growth of the TtL strain were studied as described in Section 5, and the results are shown in Fig. 2. The tissues grew up to 8-fold of inoculum weight (0.35 g) in 6 weeks. The largest amount of monomeric tannin 6 was observed in the first week of growth, which then gradually decreased with time. The contents of the oligomers 1, 11, and 13 markedly increased in the second week, and conversely to the noticeable increase in the tissue growth, the contents of 1, 11, and 13 decreased markedly on the third week. Then they were almost stable up to the sixth week, despite the continuous increase in tissue weight. The content of 2 increased gradually in tissues with time. As 2 was found to be the main tannin in the stems and roots of the intact plant as discussed below, the time-course profile of 2 may suggest an increase in the activity of its biosyn-

O

R2

O CO OC OC

O HO

O

R1 OH OH

HO OH HO

OH

OH

OH

15: R1= H, R2= OH 16:R1= OH, R2= H Fig. 4. Structures of castalagin (15) and vescalagin (16), C-glycosidic ellagitannins from Quercus alba callus culture which were grown on high Cu2+ concentration medium (Zhentian et al., 1999).

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

on high Cu2+ concentration medium (Zhentian et al., 1999). The high Cu2+ concentration also markedly inhibited production of galloylglucoses in Cornus capitata adventitious root cultures, whereas a crude enzyme solution prepared from the same cultures mediated the formation of a biaryl linkage when mixed with galloylglucoses (Tanaka et al., 2001). The effects of Cu2+ on tannin production in cultured T. tetrandra tissues were thus examined.

0.5 Content (mg/g fr. wt)

(a)

2 11 13

(2.4)

(2.4)

To investigate the effects of Cu2+ on ellagitannin formation in cultured T. tetrandra tissues, shoots were cultured on the ordinary LS agar medium (control), containing 0.1 lM Cu2+, and on media with high Cu2+ concentrations. As shown in Fig. 5a, supplemental Cu2+ of 1 and 10 lM markedly increased the dimer 11 content, and slightly increased the dimer 2 content at the expense of monomer 6, dimer 1, and trimer 13.

(b)

(2.4)

0.4 0.3 0.2

0.6

Content (mg/g fr. wt)

1982

0.5

2 11 13

0.2

0.1

(1.2)

(2.4)

(2.5)

0 60

20 40

30 30

(2.6)

0.4 0.3

0.1 0

0

6

6 1

Content (mg/g fr. wt)

Content (mg/g fr. wt)

8 6 4 2 0

6 1

0.1

1

5 4 3 2 1 0

10

(control)

+

NH4 (mM) NO3− (mM)

CuSO4 concentration (µM)

(c) Content (mg/g fr. wt)

0.4 0.3

2 11 13

0.1

(1.1)

(2.4)

40 20

(2.2)

0.2

0 14

Content (mg/g fr. wt)

12

6 1

10 8 6 4 2 0

10

30

50

(control)

Sucrose concentration (g l -¹) Fig. 5. Effects of manipulating nutrients in the culture medium. Tissue growth (g fr. wt/test tube) is presented by the numbers shown in parentheses at the top of the graph. The bars indicate the s.e. of the means of three replicates. (a) Effects of Cu2+ on tissue growth and ellagitannin (1, 2, 6, 11, and 13) contents of cultured tissues of T. tetrandra. Shoots were cultured on LS medium with supplemental Cu2+ (1 and 10 lM). Shoots for the control group were cultured on the ordinary LS medium containing 0.1 lM Cu+2. (b) Effects of NH4+/NO3 ratio on tissue growth and ellagitannin (1, 2, 6, 11, and 13) contents of cultured tissues of T. tetrandra. Different NH4+/NO3 ratios (total of 60 mM nitrogen) were added to nitrogen source-free LS medium. (c) Effects of sucrose concentration on tissue growth and ellagitannin (1, 2, 6, 11, and 13) contents of cultured tissues of T. tetrandra. Shoots were cultured on LS media with 10, 30, 50, and 100 g l 1 sucrose. Medium with 100 g l 1 sucrose was toxic to the tissues.

1983

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

fects of sucrose on ellagitannin production in cultured T. tetrandra tissues were thus investigated. Shoots were cultured on LS medium with different (10, 30, 50 and 100 g l 1) sucrose concentrations. As shown in Fig. 5c, cultured T. tetrandra tissues on medium with 50 g l 1 sucrose showed increased accumulation of 6 (12.8 mg/g fr. wt), a slight increase in the formation of 1, and low production of 2, 11, and 13. In contrast, contents of 2, 11, and 13 were slightly affected in tissues cultured on medium with 10 g l 1 sucrose, although tissue growth and formation of 6 and 1 decreased greatly. High sucrose concentration (100 g l 1) was toxic for the tissues.

2.5.2. Effects of nitrogen sources Nitrogen sources in the tissue culture medium also affect tissue growth and ellagitannin biosynthesis (Ishimaru and Shimomura, 1991; Taniguchi et al., 1998, 2000, 2002a; Zhentian et al., 1999). Therefore, the effect of nitrogen nutrient composition in the medium on the growth and tannin production of T. tetrandra cultured tissues was investigated. As shown in Fig. 5b, an LS medium modified with respect to the NH4+/NO3 ratio (total of 60 mM nitrogen) showed that a 40 mM NH4+/20 mM NO3 combination was best for tissue growth and tannin production. Although LS medium with a high proportion of the rapid source, reduced nitrogen NH4+, was effective for enhancing tissue growth and tannin production, a medium with NH4Cl as the sole nitrogen source was toxic to the T. tetrandra cultured tissues. These results are in agreement with those reported by Taniguchi et al. (1998). The other NH4+/NO3 ratios examined were less effective for tissue growth and tannin production.

2.6. Ellagitannin profiles of regenerated, acclimatized, and intact plants Shoots subcultured on LS hormone-free medium promoted root development, accompanied by an increase in shoot length leading to regeneration. Regenerated plantlets were then subcultured four to five times on hormone-free media until they became multiplebranched well-rooted plants that could adapt to ordinary soil and climate. Although HPLC analyses of aq. Me2CO extracts of T. tetrandra cultured tissues on control LS medium with phytoregulators indicated a large accumulation of 1 and 6 (Fig. 3a), leaf extracts

2.5.3. Effects of sucrose Sucrose is the most common carbon source used in plant cell, tissue, and organ culture media, whose concentration may also influence polyphenol metabolism in cell and tissue cultures (Taniguchi et al., 1998; Bauer et al., 2004; Bahorun et al., 2005). The ef-

Content (mg/g fr. wt)

14 12 10 8

6

6

1 4

2 11

2

13 0

reg1

reg2

acl1

int1

acl 2

int2 May

Nov.

Fig. 6. Individual ellagitannin (1, 2, 6, 11, and 13) contents in regenerated, acclimatized, and intact T. tetrandra plants. Regenerated plants were harvested after 2 (reg1) and 5 (reg2) subcultures on LS hormone-free medium. Leaves were collected from acclimatized (growing on soil in pots, acl1), and intact (int1) plants of T. tetrandra at November, and again from acclimatized (growing on earth in garden, acl2) and intact plants (int2) of T. tetrandra in May. The bars indicate the s.e. of the means of three replicates.

OH

OH OH

HO CO

HO HO

OC

O H2 C

O

CO O O

HO

O

OH

CO

OH OH OH

OH OH HO

17

CO

HO HO

OH2 C

OH

O

CO O O

HO

OH O

OH

CO

HO HO

HO

CO

CO OH

HO HO

OH HO

OH

18

Fig. 7. Structures of casuarinin (17) and pedunculagin (18), the main tannins of Liquidambar formosana leaves in summer and autumn (Hatano et al., 1986).

1984

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

3. Discussion

10

Content (mg/g fr. wt)

9

6 1 2 11 13

8 7 6 5 4 3 2 1 0

leaves (int2)

stems

roots

Fig. 8. Individual ellagitannin (1, 2, 6, 11, and 13) contents in leaves, stems and roots of T. tetrandra. The bars indicate the s.e. of the means of three replicates. Tannins 6 and 13 were barely detected in the roots.

from intact T. tetrandra plants which were collected in November (int1) produced large amounts of 2, 6, and 11 (Fig. 6). Compared to the cultured tissues, the regenerated plantlets, after being subcultured on hormone-free medium for two (reg1) and five subcultures (reg2) (see Section 5.5), showed a relative increase in contents of 2 and 11 accompanied by a concomitant decrease in the contents of 1 and 6. Regenerated and acclimatized plants retained ellagitannin profiles typical of intact plants. Individual ellagitannin contents of the regenerated plantlets (reg1 and reg2), which were grown under sterile conditions, were much lower (Fig. 6) than those of leaves collected in November from intact (int1) and acclimatized (acl1) T. tetrandra plants which were grown in a garden. This agrees with involvement of tannins in plant defense mechanisms against parasites and herbivores as reported by Torssell (1983). The low tannin content (Fig. 6) in acclimatized plants (acl1) relative to intact plants (int1) may have been due to nutrient deficiencies. Acclimatized plants were grown on soil in a garden, and individual tannins in leaves from the acclimatized (acl2) and intact (int2) T. tetrandra plants were again estimated by HPLC (Fig. 6) 6 months later. Because plant materials for the last experiment were collected in early summer (May), the estimated tannin content was compared with that from an experiment conducted in autumn (November). The results (Fig. 6) indicated a marked increase in the content of the oligomeric tannins 1, 2, 11, and 13, and almost stable monomer 6 content in May. Collection of leaves of T. tetrandra in May is thus recommended whenever they are used for study of the tannin constituents. Hatano et al. (1986) reported similar seasonal changes in tannins of Liquidambar formosana leaves, in which galloylglucoses and 6 was abundant in spring, and the C–C oxidative products of 6, casuarinin (17) and pedunculagin (18) (Fig. 7), became the main tannins in summer and autumn. Additionally, the contents of tannins 1, 2, 6, 11, and 13 in aq. Me2CO extracts from stems and roots, collected on the same day in May as leaves of intact T. tetrandra plants were similarly estimated by HPLC. The HPLC analyses (Fig. 8) established a large accumulation of these tannins in T. tetrandra leaves (int2), whereas the stems and roots showed 2 as the main tannin accompanied by small amounts of the other tannins. A study on T. tetrandra leaf and stem anatomy revealed the occurrence of parenchyma cells as the main leaf component, and thickened lignified elements as the major stem component (Bercu, 2008). Thus deposition of 2 may be found in thickened lignified cells, whereas deposition of other tannins (1, 6, 11, and 13) may be mainly in the non-lignified parenchyma cells.

Exploring and utilizing plant tissue cultures to produce hydrolyzable tannins has maintained the interest of numerous investigators over many years, and the establishment of in vitro cultures for tannin-rich species from several plant families [e.g., Anacardiaceae (Taniguchi et al., 1997, 2000), Cornaceae (Yazaki and Okuda, 1989; Ishimaru et al., 1993; Tanaka et al., 2001), Euphorbiaceae (Taniguchi et al., 2002a), Fagaceae (Krajci and Gross, 1987; Tanaka et al., 1995; Scalbert et al., 1988; Zhentian et al., 1999), Hamamelidaceae (Ishimaru et al., 1992), Geraniaceae (Ishimaru and Shimomura, 1991, 1995; Yazaki et al., 1991), Melastomataceae (Yazaki and Okuda, 1990), Onagraceae (Taniguchi et al., 1998, 2002b), and Rosaceae (Motomori et al., 1995)] has been reported. While searching for an alternative source for the stable production of tamaricaceous ellagitannins, we have succeeded in establishing T. tetrandra shoot cultures capable of producing monomeric to pentameric ellagitannins. Although intact plants contain very low amounts (0.22 mg/g fr. wt) of the potent cytotoxic tannin hirtellin A (1) in autumn (int1; Fig. 6), T. tetrandra cultured tissues grown on LS medium in the dark (TtD-D; Fig. 3a) accumulated large amounts of 1 (9 mg/g fr. wt). Tannin 1 was mostly localized in the undifferentiated part (TtD-U; Fig. 3b) of the tissues (12 mg/g fr. wt; 56fold its content in leaves of the intact plant in autumn). Tissue mass and ellagitannin production of the cultured tissues were also modified by manipulating culture medium nutrients. The medium with 40 mM NH4+/20 mM NO3 had the best ratio for tissue growth and tannin production. Supplemental sucrose (50 g l 1) increased accumulation of 6 (up to 12.8 mg/g fr. wt). The influences of Cu2+ on tannin production were also noticeable. Shoots cultured on LS medium with supplemental Cu2+ (1 and 10 lM) showed marked increases in the content of 11 (4-fold its content in control cultures tissue), a slight increase in the content of 2, and a concomitant decrease in the contents of 1, 6, and 13. As laccases activity in the Acer pseudoplatanus cell suspension culture is closely related to the Cu2+ level in the culture medium, as reported by Bligny et al. (1986), Zhentian et al. (1999) has attributed the increase in production of the ellagitannins castalagin (15) and vescalagin (16) in Quercus alba callus culture with high Cu2+ concentration to the involvement of laccases in the biosynthesis of galloyl biaryl linkages. Laccases are monomeric extracellular enzymes containing four copper atoms bound to three redox sites (Kunamneni et al., 2008). Recently, the involvement of laccases in ellagitannin biosynthesis was demonstrated practically by purifying two related enzymes, pentagalloyl:O2 oxidoreductase, and tellimagrandin II:O2 oxidoreductase, from Tellima grandiflora leaves that regio- and stereospecifically catalyzed the oxidation of pentagalloylglucose to 6, and oxidation of the latter into the dimeric tannin cornusiin E, respectively (Niemetz and Gross, 2003a,b). Taken together, laccases may thus be involved in the biosynthesis of tamaricaceous ellagitannins. At a 0.1 lM Cu2+ level of the ordinary LS medium, most likely, the enzyme that catalyzes the oxidation of pentagalloylglucose into 6 and the one that catalyzes the formation of the m-GOG linkage are activated. Subsequently, biosynthesis of m-GOG linear-type ellagitannins (e.g., 1, 13, and 14) through C–O oxidative coupling of a corresponding number of molecules of 6 is mostly operable (Scheme 1, route A). Alternatively, at high culture medium Cu2+ levels (1 and 10 lM), the enzymes that catalyze formation of the hellinoyl (m-GO-m-GOG) and the p-GOG linkages are activated. The suggested biosynthesis of linear tannins with a p-GOG linkage, such as 9, through C–O oxidative coupling of a corresponding number of molecules of 6 is shown by route B in Scheme 1. Biosynthesis of hellinoyl-

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

type tannins such, as 2 from 6, is shown by route E in Scheme 1. Macrocyclic tannins, such as 11 and 10, may be biosynthesized through further C–O oxidative couplings at 1 and 9, as shown by routes C and D in Scheme 1, respectively. In summary, in relation to the ellagitannin biosynthesis shown by Niemetz and Gross (2003a,b), a group of regio- and stereospecifically Cu2+ enzymes

1985

(laccases) may be involved in biosynthesis of different types of tamaricaceous ellagitannins. A slight increase in the formation of 2 (Fig. 5a) in tissue cultured in a high Cu2+ concentration medium may have been due to a low proportion of the lignified cell depositing 2 in the tissues. The content of 2 increased gradually with tissue development (Fig. 2) and was highly elevated in stiff undifferentiated tissues (Fig. 3b). This tannin was also the main

Scheme 1. In vitro oxidation of 1,2,3,4,6-pentagalloylglucose into 6, and the biosynthetic relationships of ellagitannins in cultured shoots of T. tetrandra.

1986

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

tannin in woody roots and stems of the intact plant (Fig. 8). Deposition of 2 in lignified cells was suggested, as T. tetrandra stems and roots are mainly constructed from lignified tissue (Bercu, 2008). Ellagitannins, castalagin (15) and vescalagin (16), are accumulate highly in the wood of Quercus robur and Q. petraea (Masson et al., 1994). The large accumulation of 2 in lignified tissues may thus be due to a higher incidence of biosynthetic enzyme activity. Enhanced production of the primary ellagitannin metabolite 6 in shoots (TtL-S and TtD-S; Fig. 3b) was also characteristic for the site of ellagitannin biosynthesis, which is assumed to be in the cell walls of the cells of the leaf mesophyll tissue, based on the results of an immunohistochemical study conducted by Grundhöfer et al. (2001), and the large accumulations of tannins in leaves rather than stems and roots (Fig. 8) of the naturally growing T. tetrandra plant. In contrast, cultures grown in the dark showed a large accumulation of the oligomers 2, 6, 11, and 13 (Fig. 3a) with noticeably large localization of the tannin in the undifferentiated part of the cultures (Fig 3b); the dark condition, however, showed no effect on tannin accumulation in roots of the naturally growing plant, as demonstrated from the comparable tannin contents with those of the stems (Fig. 8). Moreover, cutting the tissues during subculturing was always accompanied by the formation of such undifferentiated parts at the exposed surface of the shoots, and this transformation was always observed in all cultures. Because of the stiffness of the undifferentiated part, it has been assumed to be composed mainly of thickened lignified cells. Additionally, recent studies have suggested involvement of plant laccases in maintenance of cell wall structure and integrity, in responses to salt stress, in wound healing, in phenolic/flavanoid metabolism, as well as lignin biosynthesis, and the enzymes have also been considered to play indirect roles in the plant defense to herbivores or pathogens (Mayer and Staples, 2002; Sharma and Kuhad, 2008; Wang et al., 2008). Thus, the localization of tannins in the undifferentiated parts of the cultures may be attributable to the higher incidence of laccase activity that subsequently mediates higher tannin formation and lignin biosynthesis in these transformed tissues. Induction of undifferentiated tissues in cultures by tissue incision during subculturing process may be a physiological response similar to gall formation common to intact Tamarix plants. Indeed, Ishak et al. (1972a,b) reported that 47.94% of the total weight of Tamarix aphylla gall is tannins.

4. Conclusions Although several in vitro tannin production cultures have been established previously, few were shoot cultures. We established T. tetrandra shoot cultures that produced different types of ellagitannins common to intact tamaricaceous plants. It is the first in vitro culture that has produced tetrameric and pentameric ellagitannins. T. tetrandra cultured shoots accumulated a high amount of 1 (40fold its content in leaves of the intact plant in autumn), which had significant host-mediated antitumor activity in a previous study (Miyamoto et al., 1993) and prominent selective cytotoxic effects against a broad spectrum of tumor cell lines in our recent investigation (Orabi et al., 2010b). Supplemental Cu2+ increased production of 2 and 11 at the expense of 1, 6, and 13, suggesting a concomitant variation in biosynthetic enzyme(s) activity. Thus, the shoot culture established in our study is useful not only as a stable source for tamaricaceous tannin production but also as an aseptic, well controlled plant model system to study the biochemistry of these tannins, and may be useful for studying laccase function in plants as well. The development of a practical method for in vitro propagation of T. tetrandra shown here is advantageous, because it offers large-scale multiplication in a limited space within a short time and may be a rapid introduc-

tion of new clones with desirable ornamental features combined with more salinity tolerance. 5. Experimental 5.1. General experimental procedures The NMR instrument, analytical normal- and reversed-phase HPLC conditions, and the chromatographic gels used in this study were the same as cited in our previous paper (Orabi et al., 2009). Preparative reversed-phase HPLC was performed at 40 °C on a YMC-Pack ODS-A A-324 (YMC, Tokyo, Japan) column (i.d., 10  300 mm) using 0.01 M H3PO4–0.01 M KH2PO4–MeOH [either 2:2:1 (solvent I) or 3:3:2 (solvent II), at a flow rate of 2 ml/min, with detection at 280 nm UV]. 5.2. Plant material and culture methods Plant materials for in vitro cultures were obtained from young T. tetrandra trees (70–90 cm height) cultivated in the medicinal plant garden of the Faculty of Pharmaceutical Sciences, Okayama University. A voucher specimen (TTO-00704) was deposited in the herbarium of the same garden after being further confirmed by Prof. Susumu Terabayashi, Laboratory of Medicinal Resources, Yokohama College of Pharmacy. Actively growing branches of the new growth were excised in April 2007. The branches were cut into 5-cm portions and washed under running tap H2O for 1 h. Plant material was surface-disinfected in EtOH–H2O (70:30, v/v) for 10 s and an aq. solution of 1.0% NaOCl supplemented with 0.5% Tween 80 for 15 min, followed by two rinses in sterile distilled H2O. Stem explants, 1–2 cm long with several nodes, were placed in glass tubes (1.8  18 cm; Iwaki, Tokyo, Japan) containing 10 ml LS medium supplemented with 30 g l 1 sucrose, 10 g l 1 agar and six auxin/cytokinin combinations; 2.13 mg l 1 IAA/2.25 mg l 1 BA, 2.13 mg l 1 IAA/2.15 mg l 1 KIN, 1.86 mg l 1 naphthalene acetic acid (NAA)/2.25 mg l 1 BA, 1.86 mg l 1 NAA/2.15 mg l 1 KIN, 2.21 mg l 1 2,4-dichlorophenoxyacetic acid (2,4-D)/2.25 mg l 1 BA, or 2.21 mg l 1 2,4-D/ 2.15 mg l 1 KIN and incubated at 25 °C in the dark. Media with IAA/BA and IAA/KIN combinations were also incubated under a 12 h/day photoperiod (white fluorescent lamps, 3000 l) at 25 °C. Multiple shoot cultures were induced on basal medium supplemented with combinations of IAA/KIN or IAA/BA under light and dark conditions, and the resulting cultures consisted of undifferentiated stiff tissue touching the solid medium and multiple shoots with several leaves. Other hormone combinations promoted callus development. Because the best shoot proliferation results were obtained with the IAA/BA combination, the initiated shoots were maintained by routine subculture on 30 ml LS medium in a 100ml Erlenmeyer flask under illumination (12 h/day, 3000 l) at 25 °C every 30 days for 1 year to establish the T. tetrandra light (TtL)-grown strain. Similarly, the shoots initiated under dark conditions on the same medium were also routinely subcultured on 30 ml LS medium in 100-ml Erlenmeyer flasks at 30 day intervals for 1 year to establish the T. tetrandra dark (TtD)-grown strain. The plant materials used for the experiments shown in Figs. 6 and 8 were as follows. Regenerated plantlets (reg1 and reg2) were obtained from cultured shoots after being subcultured two (reg1) and five (reg2) times on hormone-free LS medium, as explained below. Leaves were collected in November from acclimatized plants grown in pots (acl1) and later in May from the same acclimatized plants after being grown on soil in a garden (acl2). Leaves from intact T. tetrandra plants were also collected in November and May. Because T. tetrandra leaves are very small, and scale-like, we used young branches of new growth, composed majorly of leaves on

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

green tender young stems (2–5 cm long). Different plant materials for the experiment shown in Fig. 8 [i.e., leaves (int2), stems (5– 7 mm in diameter from old growth), and roots (7–10 mm in diameter)], were collected in May from 3 years-old, actively growing T. tetrandra trees. 5.3. Isolation of tannins from cultured tissues Fresh tissues (980 g) from dark-grown cultures, which were harvested 30 days from the inoculum time, were homogenized in H2O–Me2CO (30:70, v/v, 12 l) at room temperature. The filtered homogenate was concentrated in vacuo to 500 ml at temperature below 40 °C. The concentrated homogenate was applied to a Diaion HP-20 (i.d., 5  50 cm) column eluted with H2O, MeOH–H2O (50:50, v/v), MeOH, and Me2CO, successively, to give the corresponding H2O (19.2 g), MeOH–H2O (50:50) (12.6 g), MeOH (0.61 g), and Me2CO (0.13 g) fractions, respectively. The respective fractions were fractionated by monitoring normal- and reversedphase HPLC. The H2O–MeOH (50:50, v/v) fraction (12.6 g) was subjected to a Toyopearl HW-40 (coarse grade, i.d., 2.2  60 cm) column and eluted with EtOH–H2O (50:50 ? 70:30, v/v), EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (90:10 ? 80:20 ? 70:30 ? 50:50, v/v), and Me2CO–H2O (70:30, v/v), collecting 1000-drop fractions and yielding Toyopearl fractions (Tfrs) 1–700 and the Me2CO– H2O (70:30, v/v) fraction. The Tfrs 34–40 (848 g) eluted with H2O–EtOH (50:50, v/v) was applied to a Sephadex LH-20 (i.d., 2.2  20 cm) column with EtOH– H2O (50:50 ? 70:30, v/v), and Me2CO–H2O (70:30, v/v), as eluants. The H2O–EtOH (50:50, v/v) eluate afforded a pure sample (391 g) and a crude fraction (142 g) of tellimagrandin I (5) in subsequent fractions. The Tfrs 45–74 (2.475 g) eluted with H2O–EtOH (50:50, v/v) produced tellimagrandin II (6) as the main tannin in the reversed-phase HPLC analysis with solvent 1 (tR 18 min). The Tfr 61–74 (843 mg) was subjected to an MCI-gel CHP-20P (i.d., 1.1  36 cm) column with H2O–MeOH (95:5 ? 90:10 ? 80:20 ? 75:25 ? 70:30, v/v) and MeOH. The early eluate with the H2O–MeOH (75:25, v/v) produced a crude fraction from 6 (301.5 mg), whereas the H2O–MeOH (75:25, v/v) late eluate and the H2O–MeOH (70:30, v/v) eluate yielded a pure sample of 6 (69 mg). A part (600 mg) of the Tfrs 101–201 (3.242 g) eluted with EtOH–H2O (70:30, v/v) was subjected to a Sephadex LH-20 (i.d., 2.2  20 cm) column with EtOH–H2O (50:50 ? 70:30, v/v), and Me2CO–H2O (70:30, v/v) as eluants. The EtOH–H2O (70:30, v/v) late eluate afforded a pure sample of hirtellin A (1) (111 mg). The Tfrs 202–275 (1.232 g) eluted with EtOH–H2O (70:30, v/v) was subjected to an MCI-gel CHP-20P (i.d., 1.1  36 cm) column with H2O–MeOH (90:10 ? 85:15 ? 80:20 ? 75:25 ? 70:30, v/v) and MeOH. The eluate with H2O–MeOH (85:15) (215 mg) was rechromatographed on the same MCI gel column with H2O–MeOH (90:10 ? 85:15 ? 80:20 ? 75:25, v/v) and MeOH. The H2O–MeOH (80:20, v/v) eluate was divided into two subsequent fractions (18.4 and 56.8 mg). Preparative HPLC purification of these fractions with solvent I afforded pure samples of nilotinin M2 (7) (5.3 mg), remurin A (4) (12.8 mg) and tamarixinin A (3) (15 mg), respectively. The H2O–MeOH (75:25, v/v) eluate and the early H2O–MeOH (70:30, v/v) eluate afforded another pure sample of hirtellin A (1) (282 mg), whereas the late H2O–MeOH (70:30, v/v) eluate produced an impure fraction of 1 (261 mg). A part (414 mg) of the Tfrs 276–414 (949 mg) eluted with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (90:10, v/v) was subjected to an MCI gel CHP-20P (i.d., 1.1  36 cm) column with H2O–MeOH (90:10 ? 85:15 ? 80:20 ? 75:25 ? 70:30, v/v) and MeOH. The H2O–MeOH (75:25, v/v) eluate afforded nilotinin

1987

D3 (8) (18 mg). A part (17.6 mg) of the early H2O–MeOH (70:30, v/v) eluate (68.1 mg) afforded hirtellin B (2) (4 mg) after preparative HPLC purification with solvent I, whereas the H2O–MeOH (70:30, v/v) late eluted fraction produced another pure sample of 1 (131 mg). The Tfrs 443–564 (1.1 g) eluted with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (80:20, v/v) was subjected to a Sephadex LH-20 (i.d., 1.1  21 cm) column and eluted with EtOH–H2O (70:30, v/v), EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (90:10, v/v), and Me2CO–H2O (70:30, v/v), successively, collecting 1000-drop fractions and yielding Sephadex fractions (Sfrs) A–D. Sfr-A (118 mg) eluted with EtOH–H2O (70:30, v/v) was subjected to an MCI gel CHP-20P (i.d., 1.1  36 cm) column with H2O–MeOH (75:25 ? 70:30, v/v) and MeOH. The early eluate with H2O–MeOH (70:30, v/v) afforded an additional sample of 1 (11 mg), whereas the latter fractions (8 and 19.6 mg) yielded pure samples of isohirtellin C (12) (2 mg), and hirtellin C (11) (5.3 mg) and 1 (2 mg), respectively, after preparative HPLC with solvent I. The Sfr-B (217 mg) eluted with EtOH–H2O (70:30, v/v), was subjected to an MCI gel CHP-20P (i.d., 1.1  36 cm) column with H2O–MeOH (80:20 ? 75:25 ? 70:30 ? 65:35, v/v) and MeOH. A preparative HPLC purification of part (48 mg) of the H2O–MeOH (65:35, v/v) and MeOH eluates (94.7 mg) with solvent II yielded a pure sample of 1 (7.8 mg) and tamarixinin C (9) (20.8 mg). The Sfr-C (323 mg) early eluate with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (90:10, v/v) showed a mixture of several dimeric and trimeric tannins as indicated from their retention times in normal-phase HPLC analysis (Okuda et al., 1989), whereas the late eluate with EtOH– H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (90:10, v/v), the SfrD (435 mg), was subjected to an MCI gel CHP-20P (i.d., 1.1  36 cm) column with H2O–MeOH (75:25 ? 70:30 ? 65:35, v/v) and MeOH. The eluate with H2O–MeOH (70:30, v/v) afforded pure sample of hirtellin T1 (13) (32.6 mg) and an impure fraction (78.6 mg). A preparative HPLC purification for part (40 mg) of the latter fraction gave an additional pure sample of 13 (18.8 mg) and a pure sample of tamarixinin B (10) (5.2 mg). The Tfrs 565–611 (390 mg) eluted with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (70:30, v/v) was subjected to Sephadex LH-20 (i.d., 1.1  21 cm) columns, and eluted with EtOH–H2O (70:30, v/v), EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (90:10 ? 80:20, v/v), and Me2CO–H2O (70:30, v/v), successively. The late eluate with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (80:20, v/v) (61 mg), which revealed a bundle of peaks at tR 22 min on normal phase HPLC analysis, yielded pure samples of hirtellin Q1 (14) (9 mg) upon preparative HPLC purification with solvent I. The Tfrs 612–700 (220 mg) eluted with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (50:50, v/v) was subjected similarly to Sephadex LH-20 (i.d., 1.1  21 cm) columns, and eluted with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (80:20 ? 70:30, v/v) and Me2CO–H2O (70:30, v/v), successively. The eluate with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (80:20, v/ v) (23 mg) was purified by preparative HPLC with solvent I, and yielded another pure sample of 14 (5.3 mg), whereas both the eluate with EtOH–H2O (70:30, v/v) – Me2CO–H2O (70:30, v/v) (70:30, v/v) (50 mg) and the eluate with Me2CO–H2O (70:30, v/v) showed two bundles of peaks at retention time corresponding to ellagitannin tetramers (tR 22 min) and pentamers (tR 32 min) on normal phase HPLC analyses. Normal phase HPLC analysis of the Me2 CO–H2O (70:30, v/v) fraction (41 mg) of the Toyopearl column also revealed a bundle of peaks at tR 32 min. 5.4. Estimation of the effects of nutrients and culture conditions The basal LS medium (inorganic salts and vitamins) supplemented with 10 g l 1 agar, 30 g l 1 sucrose, 2.13 mg l 1 IAA, and

1988

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989

2.25 mg l 1 BA was regarded as a control medium in this study. For studying effects of nutrients and culture conditions on the tissue growth and tannin production, tissues of the control and test groups were obtained, other than specified, by inoculating shoots (inoculum size 350 mg) from the TtL strain to 10 ml of the control LS medium (control group), and the LS medium was specifically modified with respect to certain component(s) (test groups) in a glass test tube (1.8  18 cm) with three replicates. Cultures were incubated under illumination (3000 l, 12 h/day) at 25 °C and harvested after 30 days. For the experiment shown in Fig. 2, shoots were cultured on control LS medium as described above and harvested every 7 days. For the experiment shown in Fig. 3a, shoots from the TtL strain were cultured on the control LS medium under illumination to produce the TtL-L strain (control strain) and in dark to produce the TtL-D strain. Similarly, shoots from the TtD strain were cultured on the control LS medium in dark to produce the TtD-D strain and under illumination (3000 l, 12 h/day) to produce the TtD-L strain. The experiment was repeated twice, and the results were considered identical. The average tissue growth and tannins content of the second experiment are shown in Fig. 3a. For the experiment shown in Fig. 3b, shoots from the TtD strains were inoculated (inoculum size 1 g) on 30 ml LS control medium in 100-ml Erlenmeyer flasks with three replicates, and cultured in dark at 25 °C for 30 days. Shoots (inoculum size 1 g) from the TtL strain were treated similarly and cultured under light illumination at 25 °C for 30 days. The obtained cultures of the TtL strain consisted of stiff, greenish-black undifferentiated parts (TtL-U) and green multiple shoots (TtL-S) having several leaves. Cultures of the TtD strain consisted of reddish-black undifferentiated parts (TtD-U) and whitish shoots (TtD-S) having several leaves. The shoot and the undifferentiated parts were carefully separated from each other before quantitative analyses. For examining the effect of the Cu2+ level in the culture medium, shoots were cultured on LS media with high CuSO4 concentrations, 1 and 10 lM, whereas the control was cultured on the ordinary LS medium (containing 0.1 lM CuSO4). The experiment was repeated three times, and the results were identical. The average tissue growth and tannin content of one of these experiments are shown in Fig. 5a. For the experiment shown in Fig. 5b, different ratios of NH4Cl and KNO3 (total 60 mM nitrogen) were added to nitrogen-free LS medium. Because medium with 0 mM KNO3 and 60 mM NH4Cl was toxic to the tissues, the tannin content of the tissues was not determined. For the experiment shown in Fig. 5c, shoots were cultured on media with different sucrose concentrations (10, 30, 50, and 100 g l 1). A high sucrose concentration (100 g l 1) was also toxic to the tissues. 5.5. Regeneration and in vitro adaptation of the regenerated plantlets Shoots subcultured on hormone-free medium promoted root development accompanied by increase in the shoot length on the fourth week from the inoculum time. Once the regenerated plantlets had been transferred to fresh medium, they showed fast-growing multiple-branched aerial parts and rapidly growing roots. The plantlets were resubcultured four to five times on a hormone-free medium under illumination at 25 °C until they became complete well-developed plants. Under the same light and temperature conditions, these plants were transferred to small pots containing perlite, irrigated with tap H2O, and covered with polyethylene bags to maintain high humidity. After 1 week, the bags were gradually perforated. Four weeks later, the plants were transferred to pots containing clay and successfully adapted to the ordinary soil and climate. Six months later, these acclimatized plants were then

transferred from the pots and grown directly on soil in a garden, with continued successful growth. 5.6. Quantitative determination of ellagitannins by HPLC Cultures were carefully separated from the growth media, and quantitatively transferred to clean glass vials in which the fresh weights were determined. Crude ellagitannin extract was obtained from plant materials as follows. Fresh material was chopped, crushed finely, and extracted with H2O–Me2CO (3:7, v/v) (20 ml/ g fr. wt) for 20 min in a sonic bath. After removal of the tissue debris by centrifugation, the supernatant was evaporated to dryness at 40 °C under reduced pressure. The residue was dissolved in MeOH and centrifuged, and the supernatant was subjected to reversedphase HPLC analysis under the following conditions: YMC-Pack ODS-A A-303 column (i.d., 4.6  250 mm) eluted with 0.01 M H3PO4–0.01 M KH2PO4–MeOH (43:43:14) at a flow rate 0.85 ml/ min at 40 °C with UV detection at 280 nm. The quantity of the individual tannins (1, 2, 6, 11, and 13) were determined from the peak areas referenced with authentic samples. Acknowledgments The first author thanks the Egyptian Government (Ministry of Higher Education and State for Scientific Research) for the scholarship. The NMR instrument used in this study is the property of the SC-NMR Laboratory of Okayama University. References Abouzid, S.F., Ali, S.A., Choudhary, M.I., 2009. A new ferulic acid ester and other constituents from Tamarix nilotica. Chem. Pharm. Bull. 57, 740–742. Ahmed, A.F., Yoshida, T., Okuda, T., 1994a. Tannins of tamaricaceous plants. V: New dimeric, trimeric and tetrameric ellagitannins from Reaumuria hirtella. Chem. Pharm. Bull. 42, 246–253. Ahmed, A.F., Yoshida, T., Memon, M.U., Okuda, T., 1994b. Tannins of tamaricaceous plants. VI: Four new trimeric hydrolyzable tannins from Reaumuria hirtella and Tamarix pakistanica. Chem. Pharm. Bull. 42, 254–264. Bahorun, T., Neergheen, V.S., Aruoma, O.I., 2005. Phytochemical constituents of Cassia fistula. Afr. J. Biotechnol. 4, 1530–1540. Bauer, N., Leljak-Levanic, D., Jelaska, S., 2004. Rosmarinic acid synthesis in transformed callus culture of Coleus blumei Benth. Z. Naturforsch. 59c, 554–560. Bercu, R., 2008. Tamarix tetrandra Pall. Ex. Bieb. (Tamaricaceae) stem and leaf anatomy. Sci. Papers Fac. Agric. 40 (3), 13–16, http://agricultura.usab-tm.ro/ Volum2008.php. Bligny, R., Gaillard, J., Douce, R., 1986. Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Effects of a copper deficiency. Biochem. J. 237, 583–588. Fakir, H., 2006. Flora of Bozburun Mountain and its environ (Antalya–Isparta– Burdur, Turkey). Turk. J. Bot. 30, 149–169. Gaskin, J.F., Schaal, B.A., 2003. Molecular phylogenetic investigation of U.S. invasive Tamarix. Syst. Bot. 28, 86–95. Grundhöfer, P., Niemetz, R., Schilling, G., Gross, G.G., 2001. Biosynthesis and subcellular distribution of hydrolyzable tannins. Phytochemistry 57, 915–927. Hamzaog˘lu, E., Aksoy, A., 2009. Phytosociological studies on the halophytic communities of Central Anatolia. Ekoloji 18, 1–14. Hatano, T., Kira, R., Yoshizaki, M., Okuda, T., 1986. Seasonal changes in the tannins of Liquidambar formosana reflecting their biogenesis. Phytochemistry 25, 2787– 2789. Ishak, M.S., EI-Sissi, H.I., Nawwar, M.A.M., El-Scherbieny, A.E., 1972a. Tannins and polyphenolics of the galls of Tamarix aphylla—Part I. Planta Med. 21, 246–253. Ishak, M.S., EI-Sissi, H.I., E1-Scherbieny, A.E., Nawwar, M.A.M., 1972b. Tannins and polyphenolics of the galls of Tamarix aphylla—Part II. Planta Med. 21, 374–381. Ishimaru, K., Shimomura, K., 1991. Tannin production in hairy root culture of Geranium thunbergii. Phytochemistry 30, 825–828. Ishimaru, K., Shimomura, K., 1995. Geranium thunbergii: in vitro culture and the production of geraniin and other tannins. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry: Medicinal and Aromatic Plants VIII, vol. 33. SpringerVerlag, Berlin, pp. 232–247. Ishimaru, K., Arakawa, H., Neera, S., 1992. Tannin production in Liquidambar styraciflua callus cultures. Plant Tissue Cult. Lett. 9, 196–201. Ishimaru, K., Arakawa, H., Neera, S., 1993. Polyphenol production in cell cultures of Cornus kousa. Phytochemistry 32, 1193–1197. Krajci, I., Gross, G.G., 1987. Formation of gallotannins in callus cultures from oak (Quercus robur). Phytochemistry 26, 141–143. Kunamneni, A., Camarero, S., García-Burgos, C., Plou, J.F., Ballesteros, A., Alcalde, M., 2008. Engineering and applications of fungal laccases for organic synthesis. Microb. Cell Fact. 7, 32–49.

M.A.A. Orabi et al. / Phytochemistry 72 (2011) 1978–1989 Li, H., Tanaka, T., Zhang, Y.-J., Yang, C.-R., Kouno, I., 2007. Rubusuaviins A–F, monomeric and oligomeric ellagitannins from Chinese sweet tea and their aamylase inhibitory activity. Chem. Pharm. Bull. 55, 1325–1331. Linsmaier, E.F., Skoog, F., 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18, 100–127. Lucchesini, M., Mensuali-Sodi, A., Vitagliano, C., 1993. Micropropagation of Tamarix gallica from nodal explants of mature trees. Plant Cell, Tissue Organ Cult. 35, 195–197. Masson, G., Puech, J.-L., Moutounet, M., 1994. Localization of the ellagitannins in the tissues of Quercus robur and Quercus petraea woods. Phytochemistry 37, 1245– 1249. Mayer, A.M., Staples, R.C., 2002. Laccase: new functions for an old enzyme. Phytochemistry 60, 551–565. Miyamoto, K.-I., Nomura, M., Murayama, T., Furukawa, T., Hatano, T., Yoshida, T., Koshiura, R., Okuda, T., 1993. Antitumor activities of ellagitannins against sarcoma-180 in mice. Biol. Pharm. Bull. 16, 379–387. Motomori, Y., Shimomura, K., Mori, K., Kunitake, H., Nakashima, T., Tanaka, M., Miyazaki, S., Ishimaru, K., 1995. Polyphenols production in hairy root cultures of Fragaria  Ananassa. Phytochemistry 40, 1425–1428. Niemetz, R., Gross, G.G., 2003a. Oxidation of pentagalloylglucose to the ellagitannin, tellimagrandin II by a phenol oxidase from Tellima grandiflora leaves. Phytochemistry 62, 301–306. Niemetz, R., Gross, G.G., 2003b. Ellagitannin biosynthesis: laccase catalyzed dimerization of tellimagrandin II to cornusiin E in Tellima grandiflora. Phytochemistry 64, 1197–1201. Okuda, T., Yoshida, T., Hatano, T., 1989. New methods of analyzing tannins. J. Nat. Prod. 52, 1–31. Orabi, M.A.A., Taniguchi, S., Hatano, T., 2009. Monomeric and dimeric hydrolyzable tannins of Tamarix nilotia. Phytochemistry 70, 1286–1293. Orabi, M.A.A., Taniguchi, S., Yoshimura, M., Yoshida, T., Hatano, T., 2010a. New monomeric and dimeric hydrolyzable tannins from Tamarix nilotica. Heterocycles 80, 463–475. Orabi, M.A.A., Taniguchi, S., Yoshimura, M., Yoshida, T., Kishino, K., Sakagami, H., Hatano, T., 2010b. Hydrolyzable tannins of tamaricaceous plants. III: Hellinoyl and macrocyclic type ellagitannins from Tamarix nilotica. J. Nat. Prod. 73, 870– 879. Saïdana, D., Mahjoub, M.A., Boussaada, O., Chriaa, J., Chéraif, I., Daami, M., Mighri, Z., Helal, A.N., 2008. Chemical composition and antimicrobial activity of volatile compounds of Tamarix boveana (Tamaricaceae). Microbiol. Res. 163, 445–455. Sakagami, H., Asano, K., Tanuma, S.-I., Hatano, T., Yoshida, T., Okuda, T., 1992. Stimulation of monocyte iodination and IL-1 production by tannins and related compounds. Anticancer Res. 12, 377–388. Scalbert, A., Monties, B., Favre, J.-M., 1988. Polyphenols of Quercus calli and shoots. Phytochemistry 27, 3483–3488. Sharma, K.K., Kuhad, R.C., 2008. Laccase: enzyme revisited and function redefined. Indian J. Microbiol. 48, 309–316. Sharma, S.K., Parmar, V.S., 1998. Novel constituents of Tamarix species. J. Sci. Ind. Res. 57, 873–890. Sultanova, N., Makhmoor, T., Abilov, Z.A., Parween, Z., Omurkamzinova, V.B., Attaur-rahman, Iqbal, A.M., 2001. Antioxidant and antimicrobial activities of Tamarix ramosissima. J. Ethnopharmacol. 78, 201–205.

1989

Sultanova, N., Makhmoor, T., Yasin, A., Abilov, Z.A., Omurkamzinova, V.B., Atta-urrahman, , Choudhary, M.I., 2004. Isotamarixen, a new antioxidant and prolylendopeptidase-inhibiting triterpenoid from Tamarix hispida. Planta Med. 70, 65–67. Tanaka, N., Shimomura, K., Ishimaru, K., 1995. Tannin production in callus cultures of Quercus acutissima. Phytochemistry 40, 1151–1154. Tanaka, N., Shimomura, K., Ishimaru, K., 2001. Ellagic acid formation from galloylglucoses by a crude enzyme of Cornus capitata adventitious roots. Biosci. Biotechnol. Biochem. 65, 1869–1871. Taniguchi, S., Takeda, S., Yabu-uchi, R., Yoshida, T., Yazaki, K., 1997. Production of tannic acid by Rhus javanica cell cultures. Phytochemistry 46, 279–282. Taniguchi, S., Nakamura, N., Nose, M., Takeda, S., Yabu-uchi, R., Ito, H., Yoshida, T., Yazaki, K., 1998. Production of macrocyclic ellagitannin oligomers by Oenothera laciniata callus cultures. Phytochemistry 48, 981–985. Taniguchi, S., Yazaki, K., Yabu-uchi, R., Kawakami, K.-Y., Ito, H., Hatano, T., Yoshida, T., 2000. Galloylglucoses and riccionidin A in Rhus javanica adventitious root cultures. Phytochemistry 53, 357–363. Taniguchi, S., Uechi, K., Kato, R., Ito, H., Hatano, T., Yazaki, K., Yoshida, T., 2002a. Accumulation of hydrolyzable tannins by Aleurites fordii callus culture. Planta Med. 68, 1145–1146. Taniguchi, S., Imayoshi, Y., Hatano, T., Yazaki, K., Yoshida, T., 2002b. Hydrolyzable tannin production in Oenothera tetraptera. Plant Biotech. 19, 357–363. Tashev, A.N., Taskov, E.I., 2008. Medicinal plants of Bulgarian dendroflora. Phyto. Balcan. 14, 269–278. Torssell, K.B.G. (Ed.), 1983. Natural Product Chemistry—A Mechanistic and Biosynthetic Approach to Secondary Metabolism. John Wiley & sons Ltd., Singapore. Wang, J., Wang, C., Zhu, M., Yu, Y., Zhang, Y., 2008. Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene. Plant Cell Tissue Organ Cult. 93, 303–310. Yazaki, K., Okuda, T., 1989. Gallotannin production in cell cultures of Cornus officinalis Sieb. et Zucc. Plant Cell Rep. 8, 346–349. Yazaki, K., Okuda, T., 1990. Ellagitannin formation in callus cultures of Heterocentron roseum. Phytochemistry 29, 1127–1130. Yazaki, K., Yoshida, T., Okuda, T., 1991. Tannin production in cell suspension cultures of Geranium thunbergii. Phytochemistry 30, 501–503. Yoshida, T., Hatano, T., Ahmed, A.F., Okonogi, A., Okuda, T., 1991a. Structure of isorugosin E and hirtellin B, dimeric hydrolyzable tannins having a trisgalloyl group. Tetrahydron 47, 3575–3584. Yoshida, T., Ahmed, A.F., Memon, M.U., Okuda, T., 1991b. Tannins of tamaricaceous plants. II: New monomeric and dimeric hydrolyzable tannins from Reaumuria hirtella and Tamarix pakistanica. Chem. Pharm. Bull. 39, 2849–2854. Yoshida, T., Ahmed, A.F., Okuda, T., 1993a. Tannins of tamaricaceous plants. III: New dimeric hydrolyzable tannins from Reaumuria hirtella. Chem. Pharm. Bull. 41, 672–679. Yoshida, T., Ahmed, A.F., Memon, M.U., Okuda, T., 1993b. Dimeric hydrolyzable tannins from Tamarix pakistanica. Phytochemistry 33, 197–202. Zhentian, L., Jervis, J., Helm, R.F., 1999. C-glycosidic ellagitannins from white oak heartwood and callus tissues. Phytochemistry 51, 751–756.