Solanum Mammosum L

21
XX Solanum mammosum L. (Terong Susu): In Vitro Culture and the Production of Steroidal Alkaloids and Other Secondary Metabolites G. INDRAYANTO, R. SONDAKH, A. SYAHRANI, and W. UTAMI 1 Introduction 1.1 Biology and Distribution Solanum mammosum L (S.m.), a member of the family Solanaceae, was putatively a tropical American plant. Its distribution center appears to be Central America, extending into southern Mexico, the Antilles, and the north- ern half of South America. This plant also grows well in Java from sea level to approximately 1220m (Backer and van Den Brink 1968; Miller 1969). In Indonesia, S.m. was known as terong susu or terong tete (Van Steenis et al. 1951). S. mammosum (see Fig. 1A,C) is a herbaceous or suffruticose weedy species that may be annual, biennial, or perennial in habit. It attains a height of 1-1.5 m, with a spread that may equal its height, and has sympodial branch- ing. It is densely and persistently pilose throughout, including the inflores- cences, and strongly aculeate. Its simple leaves are alternately arranged, estipulate, prominently veined, and are approximately ovate with five to seven irregular-toothed shallow lobes. Leaf size is 10-17 cm in width and 10-25 cm in length, including the petiole (Fig. 1D). It has a contracted racemose inflores- cence which arises laterally on an internode. The hypogynous, bisexual, actinomorphic flower is borne on a concave receptacle. The prominent synsepalous calyx is persistent and composed of five equal, sublate, and basally united sepals. The sympetalous corolla consists of five pale lavender to purple, basally united petals, arranged alternately with the sepals. The androecium consists of five equal, epipetalous stamens connate at the bases of their short, relatively broad filaments and borne alternately with and adnate to the petal bases. The bilocular oblong-Ianceolate, yellow anthers gradually taper apically from their middle (Miller 1969). The plant has three varieties, which differ in their fruit shape. Normally the fruit size is 4-5 cm wide and 4-8cm long. First, it is typically pear-shaped, with unusual projection from the base of the fruit, termed mammillaries; these represent abnormal, nonfunctional styles (Fig. 1A,B). The second variety has Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, JI. Dharmawangsa dalam, Surabaya 60286, Indonesia Biotechnology in Agriculture and Forestry, Vol. 41 Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj) © Springer. Verlag Berlin Heidelberg 1998

Transcript of Solanum Mammosum L

Page 1: Solanum Mammosum L

XX Solanum mammosum L. (Terong Susu): In Vitro Culture and the Production of Steroidal Alkaloids and Other Secondary Metabolites

G. INDRAYANTO, R. SONDAKH, A. SYAHRANI, and W. UTAMI

1 Introduction

1.1 Biology and Distribution

Solanum mammosum L (S.m.), a member of the family Solanaceae, was putatively a tropical American plant. Its distribution center appears to be Central America, extending into southern Mexico, the Antilles, and the north­ern half of South America. This plant also grows well in Java from sea level to approximately 1220m (Backer and van Den Brink 1968; Miller 1969). In Indonesia, S.m. was known as terong susu or terong tete (Van Steenis et al. 1951).

S. mammosum (see Fig. 1A,C) is a herbaceous or suffruticose weedy species that may be annual, biennial, or perennial in habit. It attains a height of 1-1.5 m, with a spread that may equal its height, and has sympodial branch­ing. It is densely and persistently pilose throughout, including the inflores­cences, and strongly aculeate. Its simple leaves are alternately arranged, estipulate, prominently veined, and are approximately ovate with five to seven irregular-toothed shallow lobes. Leaf size is 10-17 cm in width and 10-25 cm in length, including the petiole (Fig. 1D). It has a contracted racemose inflores­cence which arises laterally on an internode. The hypogynous, bisexual, actinomorphic flower is borne on a concave receptacle. The prominent synsepalous calyx is persistent and composed of five equal, sublate, and basally united sepals. The sympetalous corolla consists of five pale lavender to purple, basally united petals, arranged alternately with the sepals. The androecium consists of five equal, epipetalous stamens connate at the bases of their short, relatively broad filaments and borne alternately with and adnate to the petal bases. The bilocular oblong-Ianceolate, yellow anthers gradually taper apically from their middle (Miller 1969).

The plant has three varieties, which differ in their fruit shape. Normally the fruit size is 4-5 cm wide and 4-8cm long. First, it is typically pear-shaped, with unusual projection from the base of the fruit, termed mammillaries; these represent abnormal, nonfunctional styles (Fig. 1A,B). The second variety has

Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, JI. Dharmawangsa dalam, Surabaya 60286, Indonesia

Biotechnology in Agriculture and Forestry, Vol. 41 Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj) © Springer. Verlag Berlin Heidelberg 1998

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Fig. lA-E. S. mammosum, general appearance of plant with mature fruits (A); pear-shaped mature fruit with mammillaries (8); plant with flowers and immature fruits (C); mature leaf and flowers (D); long section of the mature fruit and seeds (E)

a pear-shaped fruit which has no mammilaries. A third form has spherical­shaped fruits. The fruits of different varieties do not differ significantly in their sola so dine content (Telek et al. 1977). The seed develops from an anatropous ovule and has an incumbent type of cotyledon arrangement. The coiled em­bryo is completely enclosed by a copious fleshy endosperm. The mature seeds are oval-semi orbicular in outline, flattened laterally, with the hilar end often slightly more flattened, aprroximately 3-3.5mm wide, 4-4.5mm long, and 1mm thick. They are nonglossy, reddish brown to dark brown in color, with a finely reticulate surface (Miller 1969; see Fig. 1E). The plant is tolerant of many soil types, including heavy clays. It resists drought, excessive rainfall, and mild inundation, and is fairly immune to insect attacks (Telek et al. 1977).

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396

Solasodine

Tomatidinol

Sterol

Cholesterol

Campesterol

Sitosterol

Stigmasterol

R v

R=

~ A

~ A

yvt( yvt(

A

G. Indrayanto et al.

Diosgenin

Tomatidine

Fig. 2. Structure of solasodine and its epimer, diosgenin, and sterols of the callus cultures of s.m.

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1.2 Phytosteroid Content in S. mammosum

Steroidal alkaloids with the complete and unaltered C27 skeleton of cholestane, showing different heterocyclic ring systems such as spirosolanes and solanidanes occur in the family Solanaceae (Groger 1988). The most important of the steroidal alkaloids is solasodine, due to its feature as the starting materials for the synthesis of the steroid drugs (Macek 1989). Gener­ally, solasodine is found as glycosides in the genus Solanum. The most promi­nent of the steroidal glycoalkaloids that accumulate in S.m., are solasonine, j3-solamargine, and solamargine (Figs. 2, 3). The solasodine content in the fruits is 0.20-1.20% dry wt., but the leaves are free of the steroidal alkaloids (Telek 1977; Telek et al. 1977; Tarigan 1980). Sawariam (1986) reported that the fruits also contain diosgenin and phytosterols. The solasodine content was found to increase in the green fruit until maturity (yellow fruit) and to de­crease rapidly as yellowing progressed (Fig. 4; Telek et al. 1977). The influence in other Solanum spp. of the degree of maturity of the fruits or leaves on their solasodine content was also reported (Carle 1979; Indrayanto et al. 1985). Therefore for commercial production of solasodine from S.m., the fruits must be picked as their color is in transition between green to yellow. According to Tarigan (1980), one 6-month-old s.m. plant cultivated in Lembang (West Java, 1200m above sea level) could produce ca. 4kg fruits, so about 46-67kg solasodine could be produced from 1 ha of these plants in 6 months. This is much higher compared to tomatidenol (C-22 epimer of solasodine) production from 1 ha of Solanum dulcamara plants, which produced only ca. 15-45 kg of the steroidal alkaloid (Ehmke and Eilert 1993). The yield large-scale extrac­tion of solasodine from 1 ha of S.m. plants in Puerto Rico was 24.1 kg. This yield compares favorably with the other Solanum spp. (aviculare, auriculatum, laciniatum, marginatum) that were evaluated in other plantations (Telek et al. 1977).

Glycoside

p-Solamargine

Solamargine

Solasonine

R = Solasodine

Sugar moiety

Rhamnose ~ (1--*4~ Glucose p (1--*3~ R

Rhamnose ~ (1--*2);> Glucose P (1--*3 ~ R

Rhamnose ~ (1--*4

Rhamnose ~ (1--*>2)

alactose P (1--*3~ R Glucose P (1--*3)

Fig. 3. Structures of steroidal glycoalkaloids that accumulated in the fruits of S.m.

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1.0

0.8

;;i ~

C 0.6 Q)

C 0 () Q) 0.4 c '5 0 rJ) co

(5 0.2 C/)

0~-----Y~----~~--~~-----4--____ ~ Green Yellow Green Ye llow Yel low Orange Orange

Fruit Color

Fig. 4. Effect of the degree of maturity (color) of S.m. fruit on its solasodine content (% dry wt.). (Data from Telek et al. 1977)

1.3 Pharmacological and Biological Activities of Phytosteroids

1.3.1 Solasodine

Steroidal glycoalkaloids and saponins that are widely distributed in Solanum spp. are thought to be very important in the natural defense of plants against microorganisms and/or predators (Paczokowski and Wojciechowski 1993).

A tumor-inhibiting activity of the glycoalkaloid ,B-solamargine was re­ported by Kupchan et al. (1965). Cham et al. (1987) showed that the glycoalkaloids solasonine, solamargine, and another glycoside containing solasodine-aglycone had antineoplastic activity against Sarcoma 180 in mice. Solasodine also showed growth inhibition activity to some fungal strains, although its activity was less compared with solaftoridine and verazine (Kusano et al. 1987a). Some steroidal alkaloids, including solasodine, showed an inhibitory effect on the enzymatic conversion of dihydrolanosterol into cholesterol (Kusano et al. 1987b). Roddick et al. (1990) reported that solasonine and solamargine had the membrane-disrupting properties of phosphatydy1choline/cholesterol at a concentration above 50,uM. Strong antiviral activity of the steroidal glycoalkaloid solasonine, tomatine, and chaconine was shown by Thorne et al. (1985) by using Herpes simplex infec­tion of in vitro-cultured cells. Potatoes containing more than 0.02% steroidal glycoalkaloids are considered to be toxic to man. The majority of steroidal glycoalkaloids in potatoes are derived from solanidine and tomatidenol (Kuc 1984). Acute toxicity was observed after oral/intraperitonia/intravenous administration of a-tomatine in rabbit and rat. The steroidal glycoalkaloid a-

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tomatine has the Sa-derivate of tomatidenol (tomatidine) as aglycone (Van Gelder 1989).

Frohne (1993) reported that solasodine had a cortisone-like effect, such as antiphlogistic and reducing blood vessel permeability. He reported also that solasodine could prevent anaphylactic shock in guinea pig. In a clinical trial, a dose of 1 mg solasodine citrate, twice given a day, showed a cardiotonic effect. Solanum dulcamara, which also contains steroidal glycoalkaloids, was used as a folk medicine in Europe, China, and Japan (Ehmke and Eilert 1993).

1.3.2 Diosgenin

Thewles et al. (1993) reported that biliary cholesterol output in rats was stimulated more than threefold by feeding with diosgenin, whereas biliary outputs of phospholipid and bile salts were not changed by diosgenin feeding. Takechi et al. (1992) showed that some synthetic diosgenyl f3-D-glucosides have hemolytic and antifungal activities.

Miles et al. (1994a,b) demonstrated that geeldikkop (plant-induced hepatogenous photosensitization diseases) could be induced in sheep by oral administration of crude saponins/extracts of Tribulus terrestris. The adminis­tered saponin was found to contain steroidal sapogenin diosgenin, yamogenin, tigogenin, gitogenin and neotigogenin.

1.3.3 Sterols

Sterols and their derivatives promote and maintain growth and development in plant and fungi by acting as membrane constituents. Experimental data showing that sterol acts as a hormone in plants, are scarce (Grunwald 1975; Burden et al. 1989).

Sitosterol is used as a hypolipidemic agent, in conjunction with dietary modification (usual dose 2-6 g orally). It is also used in prostate disorders (Reynolds 1993). Recent studies by Santos et al. (1995) showed that sterols (stigmasterol and sitosterol) had anti nociceptive action in mice. Given orally, stigmasterol and its acetate derivate exhibit significant though less potent analgesic action against both acetic acid- and formalin-induced noc­iceptive in mice. Stigmasterol, stigmasterol acetate, and sitosterol, given intraperitoneally, inhibited acetic acid-induced abdominal constriction in mice.

2 In Vitro Culture Studies

Extensive studies have been conducted on various in vitro aspects and the production of secondary metaboloites in Solanum aviculare, S. laciniatum

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(Macek 1989), S. aculeatissimum (Nabeta 1993), S. eleagnifolium (Giulietti et al. 1991), S. dulcamara (Ehmke and Eilert 1993), and S. glaucop­hyllum (Weissenberg et al. 1993). Our work on S. mammosum is discussed here.

2.1 Callus Cultures

In our laboratory, the callus cultures of S.m. (cell line sm) was first initiated by Isnaeni (1986) from young leaf petioles of a 6-month old plant, collected on a mountain in Nongkojajar Pasuruan East Java. The explants were cultivated on modified MS (Murashige and Skoog 1962) medium with the addition of 2mg/1 kinetin and 1 mg/12,4-D. Callus was formed within 7-10 days of incubation. From the various combinations of kinetin and 2,4-D that have been tested for these cultures, 2 mg/l kinetin and 0.5 mg/l of 2,4-D (medium K2Do.s) gave the best growth rate. All the calli were maintained in continuous light (ca. 8001x) at 25 ± 1°C and subcultured every 3 weeks (Fig. 5A,B). Suharno (1986) reported that the callus cultures of S.m. (cell line sm) could also grow well on modified MS medium with the add­ition of 2mg/1 kinetin and 0.5mg/1 NAA (medium K2Do.s) After 1 year of subculturing, the cultures exhibited friable callus with pale yellow to cream (on medium K2DOS) or pale green (on medium K2NOS) color. These calli are still growing well after 10 years of subculturing; however, these callus cultures have a shorter lag phase in their growth curve compared to the 1-year-old calli. The calli of cell line sm could also grow well in the darkness (Fig. 6).

Using 3% sucrose, glucose, or lactose in the media, Wijono (1987) re­ported that sucrose was the best carbohydrate source for the growth of callus cultures of S.m. (cell line sm). Whereas Susilowati (1987) showed that the optimum concentration of sucrose was 3% (see Figs. 7, 8), Sarwetini (1988) demonstrated that the addition of banana powder could significantly increase the growth rate of these callus cultures.

Callus cultures of S.m. could also be initiated by using shoots from shoot cultures as explants. We recently initiated some new cell lines of the callus cultures (code: sm-1, sm-12, sm-23) by using shoots from shoot cultures (cell line sm-1, sm-12, sm-23) on medium K2Do.5' Figure 6 shows that the calli ofthe new cell line sm-1 (800Ix, 25 ± 1°C) have relatively lower growth rate com­pared to the sm cell line.

Suspension cultures of s.m. (cell line sm) used for biotransformation studies (see Sect. 2.5) could be initiated by inoculating ca. 5-10g friable calli in 50ml medium K2No.5 in a 250-ml Erlenmeyer flask. After 10-15 subcultures, cell aggregates 3-7mm in diameter were formed (see Fig. 5C,D). However, the cell aggregate suspension cultures have a very low growth rate compared to the fine cell suspension. The cells appeared to be self-immobilized by subculturing (Fig. 9). The formation of cell aggregates (self-immobilization) in suspension cultures of Solanum aviculare was also demonstrated (Tsoulpha and Doran 1991).

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Fig. SA-D. Callus cultures of S.m. cell line sm (AI, B), sm-l (A2) cultivated on medium K2NUS; Cell aggregate suspension cultures of cell line sm (C, D). All the cultures were maintained under continuous light (ca. 800 Ix) at 25 ::':: 1 °C

2.2 Shoot Cultures and Micropropagation

By using young shoots bearing three to four axillary buds from a 7-month-old S. mammosum plant collected at Purwodadi Botanical Garden Malang as explants, Isfidiati (1988) initiated shoot cultures of cell line sm-p for micropropagation. After surface sterilization with 2% NaOel solution, the

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402

14

12

.- 10 Ol --.c. 8 Ol

.~ 6 .c.

(/) (J) ....

4 LL

2

0

- "'-

0

1'~ ----~ -----}:- -----:1J;- - - - __

----'I :t:

10 20

Days 30 40

G. Indrayanto et al.

50

Fig. 6. Growth curve of callus cultures of S.m. cultivated on medium K2Do.5' The calli were maintained in continuous light at light intensity of ca. 800 Ix [sm(L) and sm-l] or in darkness (D) at 25 ± 1°C. [Data of cell line sm(L), 1985 from Isnaeni 1986]

7 -.-Sucrose

J 6 -.-Glucose I -A-Lactose 5

I x (J) "0 4 c .c. I ~ 3 0 .... (9

2

• 0 2 3 4 5

Weeks

Fig. 7. Effect of some carbohydrate sources (3 %) in medium K2Do.5 on the growth rate of callus cultures of cell line sm. (Data from Wijono 1987)

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>< Q)

7

6

5

-g 4

~ 3

c'5 2

-.- Sucrose 1 %

-.- Sucrose 3% -A- Sucrose 5%

-,,- Sucrose 7% ----+--- Sucrose 10%

o 2 3

Weeks

403

---~~--'.

4 5 6

Fig. 8. Effect of sucrose concentration in medium K,Dos on the growth rate of callus cultures of cell line sm. (Data from Susilowati 1987)

90 • Fine cells /--:--. 35

Q) • Cell aggregates • -1' '. ..--------. ..x:: (/)

E 80 / :.' '. (II

::J 30 u:: (5 -> r: § 70 • (j) • 25 .... () ~

Cl ..x:: 60 . . '(jj

0 ./ /-'-A ---:-i_A (II 20 :;:

0- j --•. ~

'::f!. or £ .,

(/)

50 Q) 0 ....

140

15 LL.

/' ... ---/.. .' .... ' ...:..-~I~~' 10

30 0 2 4 6 8 10 12

Days

Fig. 9. Effect of self-immobilization of the cells on the growth rate of suspenson cultures of S.m. (cell line sm). Data represent mean of three replicates

explants were cut into one to two nodal pieces (ca. 0.5-1 cm long) and culti­vated on modified MS media with 32 combinations of phytohormones. Shoots were formed in 2-4 weeks in ten hormone combinations (see Table 1). The optimal shoot formation was obtained by using a modified MS medium with

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Table 1. Effect of hormone combination on the number of shoots formed on the inoculated shoot of S.m. (cell line sm-p). (Data from Isfidiata 1988)

Hormone (mg/l) No.

Kinetin BAP NAA IAA 2,4-D GA3 of shoots'

4 0.1 + 4 0.5 + 0.5 2 0.5 4 0.5 0.5 0.5 1.5 2 0.5 ++ 4 0.1 4 0.5 + 4 +++

" +, Number of shoots = 2-3; + +, = 4-6; + + + = 7-10.

Table 2. Percentage of root formation on the inoculated shoots of S. mammosum (cell line sm-l) cultures

Days lEA

2.5mg/1 ('Yo) 5mg/1 ('Yo)

4 0 0 8 42 57 12 67 91 17 91 100 23 100 100

Callus Root formation formation

+ +

+ + + +

+ +

the addition of 4mg/1 kinetin as the phytohormone. Our recent studies showed that shoot multiplication of the shoot cultures of s.m. (cell lines sm-1, sm-12, and sm-23) could also be achieved by using a modified MS medium with the addition of2mg/1 BAP as the phytohormone (see Fig. 10A,B). In this case, we used sterile-seedling hypocotyls as explants for initiating shoot cultures.

Pranachita (1992) reported that induction of root formation on the inocu­lated shoots of cell line sm-p could be achieved by using a modified MS medium with the addition of 0.5 mg/l IAA as phytohormone. She demon­strated that 90% root formation on the inoculated shoots resulted within 2-3 weeks.

Our recent studies showed that root induction of the cell line sm-1 shoot cultures was also successful on using a modified MS medium with the addition of 2.5 mg/l or 5 mg/l IBA as hormone; 100% root formation on the inoculated shoots occurred within 14-16 days (Table 2, Fig. lOD).

About 85% of the plantlets of cell line sm-1 survived and grew well after transplanting to common trays containing a sterilized mixture of sand and humus (1: 1) (Fig. 10C). After acclimatization for 3-4 weeks, the plants can be cultivated in the field.

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Fig. lOA-D. Shoot cultures of cell line sm-l on modified MS medium with the addition of 2mg! I BAP. One-week (A) and 4-week (8) cultures after subculturing. Young plant of SM (cell line sm-1),3 weeks after transplanting onto a glass tray containing a mixture of sand and humus (1: 1) for acclimatization (C). Root formation on the inoculated shoot of cell line sm-l after 3 weeks of cultivation on modified MS medium with the addition of 2.5 mg!1 IBA (D)

2.3 Phytosteroid and Triterpenoid Content in Tissue Cultures

Indrayanto et al. (1986) reported that callus cultures of cell line sm cultivated on modified MS medium (K2DOS) contained only cholesterol, campesterol, stigmasterol, and sitosterol, whilst solasodine and diosgenin could not be detected. Our unpublished results also showed that our new cell line of callus cultures, sm-l, sm-12, and sm-23, cultivated on media K2No.s, also could not produce solasodine. The absence of solasodine in undifferentiated cells is reported in many publications, e.g., in calli of Solanum laciniatum, S. wrightii, S. khasianum, S. aviculare. S. aculeatissimum, and S. aculeastrum (Carle 1979;

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Indrayanto et al. 1983; Galanes et al. 1984; Nabeta 1993; Drewes and Staden 1995).

Attempts to induce solasodine formation in these callus cultures by addi­tion of various concentrations of yeast extracts (Mufidah 1988) and Rhizopus arrhizus (Karsana 1988) as bioelicitors failed. In the last two experiments, the phytosterol contents were also not changed significantly. UV irradiation (21 W/m2, 24h) of cell line sm-1 calli also could not stimulate the production of solasodine, although the same UV radiation could double the hecogenin con­tent in the callus cultures of Agave amaniensis (Rusli 1996). The phytosterol (mostly sitosteol) content in callus cultures of cell lines sm and sm-1 was 0.4-0.7 mg/g drywt.

Although solasodine was not detected in callus cultures of Solanum laciniatum, as described above, Indrayanto et al. (1995) showed that its shoot cultures could produce solasodine, so it seemed that solasodine production was correlated with the availability of chlorophyll and cell differentiation, as reported in many publications (e.g., Conner 1987; Ehmke and Eilert 1993). However, our recent studies showed that in all of our shoot cultures (cell lines sm-p, sm-1, sm-12, sm-23) of S.m., solasodine was not detected. These studies showed that the production of solasodine in the plant cells was not dependent on the cell differentiation or the availability of chlorophyll in the cells. A recent publication by Ripperger (1995) reported that solasodine could also be isolated from roots of various Solanum spp. According to Subroto and Doran (1994), the hairy root cultures of Solanum aviculare produced solasodine at a concentration of 29-32mg/g dry wt. These results confirmed that the biosynthesis of solasodine might not be correlated to the availability of chloro­phyll in some Solanum species.

Recently, cell line sm also produced betulin, a lupane (triterpenoid) de­rivative (identified by TLC, MS). Now we are in the process of identifying three other triterpenoids that are isolated from the chloroform extract of these calli. The production of some lupane derivatives (betulinic acid, betulin, lupeol, and lupeol aldehyde) in callus cultures of Solanum laciniatum, S. wrightii (Indrayanto et al. 1983) and S. aviculare (Vanek et al. 1985) was also reported (see Fig. 11). Betulinic acid concentration in calli of Solanum aviculare was relatively very high (up to 3 % dry wt.).

2.4 Biotransformation by Using Suspension Cultures

Using callus cultures of cell line sm, Sondakh (1989) reported that progester­one added as substrate into the media could be transformed to 5a-pregnan-3, 20 dione (Fig. 12). The transformation of progesterone to 5a-pregnan-3,20 dione was also reported from various tissue cultures systems (Furuya et al.; 1971. Stohs and Rosenberg, 1975).

Our recent studies showed that suspension cultures of cell line sm could also transform some salicylate derivatives (salicyl alcohol, salicylic acid, and salicylamide into their mono glucoside (see Fig. 12). A new bioconversion product, salicylamide 2-0-f3-D-glucopyranoside, was isolated from the cell

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Fig. 11. Structure of some lupane derivatives accumulated in Solanum spp. tissue cultures

Lupane - derivative

Lupeol

Betulin

Betulin aldehyde

Betulinic acid

407

R

Cn3 Cn20n

cno

coon

suspension cultures of S.m. (cell line sm) following the administration of salicylamide (Syahrani et al. 1997).

The glucosylation capability of cell suspension cultures of cell line sm reported here, namely, 51.8% (salicyl alcohol) and 81.9% (salicylamide) con­version of the administrated substrates to the monoglucosides, was higher than that reported previously for other suspension cultures (Mizukami et al. 1983; Dombrowski 1993). These results showed that the suspension cultures of S. mammosum could be used to transform exogenous substrates to their glycosides (Syahrani 1997).

2.5 Antifertility Effect of Callus Cultures

Rahayu (1988) showed that the petroleum ether (40-60 0C) extract (1 mg extract equivalent with 48mg dried calli) of callus cultures of cell line sm given orally, have a significant antifertility effect in mice (Table 3). The extract (1-4mg/30g mice) did not appear to affect the behavior or activity ofthe treated mice.

The acetone extract of the same calli (1 mg extract equivalent with 46.5 mg dried calli) also exhibited a significant antifertility effect in mice at a dose of 2-4mg/30g mice (Table 3; Samesti 1988).

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cm 1 C=O

Progesterone 5 -pregnan-3. 20-dionc

--Salicylamide Salicylamide 2-0-11-glucollyranoside

Salicyl alcohol Salicin

hJH l°°~H C(x)H 1"0-( rQJ ~ if CH20H

Salicylic acid Salicylic acid glucoside

Fig. 12. Biotransformation of progesterone, salicylamide, salicyl alcohol, and salicylic acid by suspension cultures of S.m. (cell line sm)

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Table 3. Antifertility effect of S.m. (cell line sm) callus cultures

Group Extract No. of Total of Average of Dose of extracts females litters litters/mice (mg)'

Control 18 215 12 0 T1 Petroleum 18 199 11 1 T2 ether" 18 59 3 2 T3 18 16 1 4

Control 6 56 9 0 T5 Acetone" 6 8 1 2 T6 6 0 0 4

, Data from Rahayu (1988). " Data from Samesti (1988). , Dose for 30-g mice.

It was postulated that the triterpene content of these calli might cause the antifertility effect in these experiments. It is well known that many triterpenes have some cytotoxic activities (Das and Mahato 1983).

3 Conclusion

In vitro cultures of Solanum mammosum were initiated on modified MS medium with the addition of 2 mg/l kinetin and 0.5 mg/l 2,4-D or 2 mg/l kinetin and 0.5mg/1 NAA (for callus cultures), 2mg/1 BAP (for shoot cultures); 100% root formation occurred on the inoculated shoots by using a modified MS medium with the addition of 2.5 or 5 mg/l BAP.

Although callus cultures could produce only some sterols and lupane derivatives, these cultures could transform progesteron and some salicylate­derivatives which were added as substrates. Solasodine was not detected in all tissue cultures of S.m.

The petroleum ether and acetone extract of callus cultures showed a significant antifertility effect in the treated mice. This activity might be due to the triterpene content of the calli.

4 Protocols for Tissue Culture

4.1 Callus Cultures

Method 1. The explants (young leaf petioles) were washed with distilled water, ethanol 95%, and surface sterilized in 2-3% sodium hypochlorite for 4-6 min, then washed three times with sterile distilled water. The leaf petioles cut ca. 0.5-1 cm long and placed on modified MS (Murashige and Skoog 1962) medium with the addition of 2 mg/l kinetin, 0.5 mg/12,4~D, 3% sucrose, and 0.8% agar (medium K,Du,). then incubated in continuous light (ca. 800 Ix) at 25 ± 1°C.

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Method 2. Sterile shoots of the shoot cultures were cut ca. 0.5-1 cm long, and placed on the medium K2Do.5• Incubated as in method l.

The established callus cultures must be subcultured every 3-4 weeks.

4.2 Suspension Cultures

Transfer 5-10 g soft friable calli into 50 ml modified MS medium with 2 mg/l kinetin, 0.5 mg/l NAA, 3% sucrose, (medium K2DOS) in 2501. Erlemeyer flasks. Shake at 100rpm on a gyratory shaker under the same conditions as callus cultures; subculture every 7 days.

4.3 Shoot Cultures

For initiating shoot cultures, sterile seedling hypocotyl explants or surface sterilized (2-3% NaCIO) young shoots of in vivo plants, cut 0.5-1 cm long (with one to two nodal pieces) were used as explants. Modified MS medium with 2mg/1 BAP or 4mg/1 kinetin, 3% sucrose, 0.8% agar was used for initiating and maintaining the shoot cultures. The shoot cultures were incubated in ca. 1500-2000 Ix at 25 :+:: 1 dc. Subculture every 3-4 weeks.

4.4 Measurement of Growth Index (GI)

The GI of the in vitro cultures was calculated as final/initial fresh weight.

4.5 Quantitative Analysis of the Phytosteroids

Extraction. One g of powdered dried biomass was extracted three times using a vortex mixer (15 min) with 7.5 ml chloroform. All the extracts were combined and evaporated under N2 to dryness. This chloroform extract contains free sterols and triterpenes.

The residue was hydrolyzed with 2N HCI in methanol (2h, 75-80°C), neutralized with ION NaOH, then diluted with 25 ml water, and the steroidal alkaloids and sapogenins were extracted three times with 10ml chloroform. The CHCl3 phase was collected and evaporated to dryness under N2•

4.6 Analysis of Sterols and Lupane Derivatives

Sterols and lupane derivatives can be separated and determined by gas chromatography according to Indrayanto (1983) using a glass column (2m X 2mm) containing 3% OV-1 on Gaschrom Q 100-120 mesh under the following conditions: oven temperature is programmed from 220 to 280°C at 4°C/min; FID and injector temperature are 300°C, the flow of carrier gas helium is 30 mll min. By this GC method, squalene, cholesterol, campesterol, stigmasterol, sitosterol, lupeol, lupeol aldehyde, and betulin are well separated.

For determining the total sterols in the biomass, a densitometric method using Kieselgel 60 precoated plates (Merck) and CHCl3 : EtOAc (4: 1) as eluents can be used (Indrayanto et al. 1993). Quantitation was done by measuring the absorbance reflectance of the sterol spots after visualizing with anise aldehyde-H2S04 reagent (at 395 nm).

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4.7 Analysis of Solasodine

Solasodine in the biomass can be determined by using a densitometric method according to Indrayanto et al. (1995). Kieselgel 60 pre coated plates (Merck) are used as stationary phase. As eluent, a mixture of CHCI3 : MeOH : diethylamine (20: 2: 0.5) is used. The solasodine spots were detected by anise aldehyde-H2S04 reagent. Quantitation was performed by measuring the absorb­ance reflectance at 385 nm.

4.8 Analysis of Diosgenin

Diosgenin content of the biomass can be assayed by a densitometric method according to Indrayanto et al. (1994). Kieselgel 60 precoated plates (Merck) are used as stationary phase. As eluent, a mixture of n-hexane, EtOAc (3: 1), is used. After visualizing the diosgenin spots by anise aldehyde-H2S04 reagent, quantitation is performed by measuring the absorbance reflectance at 427 nm. For determining diosgenin in the presence of solasodine a GC method according to Carle (1979) was recomended. He used a glass column containing 3% SE-30 on gaschromm Q 100--120 mesh, under the following conditions: oven temperature was 250°C isothermal, detector (FID) and injector temperature were 300°C, flow of the carrier gas (helium) was 30mUmin. When the biomass also contains the epimer of diosgenin (e.g., yamogenin) an HPLC method (Wu and Wu 1991) was suggested. This uses a Zorbax-ODS column (25mm X 4.6mm i.d.) and 94% methanol in H20 as the mobile phase, with an RID detector. The separation was carried out at low temperature (O°C).

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