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
Solanum mammosum L. (Terong Susu) 395
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 spherical-
shaped 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 em-
bryo 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).
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.
.Solanum mammosum L. (Terong Susu) 397
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). Gener-
ally, solasodine is found as glycosides in the genus Solanum. The most promi-
nent 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 de-
crease 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 extrac-
tion 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.
398 G. Indrayanto et al.
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 Yellow 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 re-
ported 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 infec-
tion 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-
Solanum mammosum L. (Terong Susu) 399
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 adminis-
tered 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 noc-
iceptive 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
400 G. Indrayanto et al.
(Macek 1989), S. aculeatissimum (Nabeta 1993), S. eleagnifolium (Giulietti
et al. 1991), S. dulcamara (Ehmke and Eilert 1993), and S. glaucop-
hyllum (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 K
2
D
o
.
s
) gave the best growth rate. All the calli were maintained
in continuous light (ca. 8001x) at 25 1C 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 add-
ition of 2mg/1 kinetin and 0.5mg/1 NAA (medium K
2
D
o
.
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) re-
ported 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 K
2
D
o
.5' Figure 6 shows that the calli ofthe
new cell line sm-1 (800Ix, 25 1C) have relatively lower growth rate com-
pared 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 K
2
N
o
.
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).
Solanum mammosum L. (Terong Susu) 401
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 ::':: 1C
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
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 K
2
D
o
.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 1C. [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)
Solanum mammosum L. (Terong Susu)
><
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 culti-
vated 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
404 G. Indrayanto et al.
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 inocu-
lated 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 demon-
strated 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.
Solanum mammosum L. (Terong Susu) 405
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 K
2
N
o
.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;
406 G. Indrayanto et al.
Indrayanto et al. 1983; Galanes et al. 1984; Nabeta 1993; Drewes and Staden
1995).
Attempts to induce solasodine formation in these callus cultures by addi-
tion 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/m
2
, 24h) of cell line sm-1 calli also could not stimulate the production of
solasodine, although the same UV radiation could double the hecogenin con-
tent 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 chloro-
phyll in some Solanum species.
Recently, cell line sm also produced betulin, a lupane (triterpenoid) de-
rivative (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 progester-
one 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
Solanum mammosum L. (Terong Susu)
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) con-
version 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).
408 G. Indrayanto et al.
cm
1
C=O
Progesterone
5 -pregnan-3. 20-dionc
--
Salicylamide
Salicylamide 2-0-11-glucollyranoside
Salicyl alcohol
Salicin
hJH
l ~ 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)
Solanum mammosum L. (Terong Susu) 409
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 salicylate-
derivatives 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 3% sucrose, and 0.8% agar
(medium K,D
u
,). then incubated in continuous light (ca. 800 Ix) at 25 1C.
410 G. Indrayanto et al.
Method 2. Sterile shoots of the shoot cultures were cut ca. 0.5-1 cm long, and placed on the
medium K
2
D
o
.
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, 75-80C), neutralized with ION
NaOH, then diluted with 25 ml water, and the steroidal alkaloids and sapogenins were extracted
three times with 10ml chloroform. The CHCl
3
phase was collected and evaporated to dryness
under N
2

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
280C at 4C/min; FID and injector temperature are 300C, 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 CHCl
3
: 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-H
2
S0
4
reagent (at 395 nm).
Solanum mammosum L. (Terong Susu) 411
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 CHCI
3
: MeOH : diethylamine (20: 2: 0.5) is used. The solasodine spots were
detected by anise aldehyde-H
2
S04 reagent. Quantitation was performed by measuring the absorb-
ance 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-H
2
S04 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 250C isothermal, detector (FID)
and injector temperature were 300C, 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 H
2
0 as the mobile phase, with an RID detector. The separation was carried out at low
temperature (OC).
References
Backer CA, Van den Brink RCB (1968) Flora of Java, vol II. Wolters-Noordhoff, Groningen, pp
470-472
Burden RS, Cooke DT, Carter GA (1989) Inhibitors of sterol biosynthesis and growth in plants
and fungi. Phytochemistry 28:1791-1804
Carle R (1979) Untersuchungen zur Steroidalkaloid- und Sapogeninfiihrung in Pflanzen und
Zellkulturen der Gattung Solanum L. PhD Thesis, University of Tuebingen, Tuebingen
Cham BE, Gilliver M, Wilson L (1987) Antitumour effect of glycoalkaloids isolated from Solanum
sodomaeum. Plant a Med 53:34-36
Conner AJ (1987) Differential solasodine accumulation in photoautotrophic and heterotrophic
tissue cultures of Solanum laciniatum. Phytochemistry 26:2749-2750
Das MC, Mahato SB (1983) Review of triterpenoids. Phytochemistry 22:1071-1095
Dombrowski K (1993) Phytochemische und enzymologische Untersuchungen zur Biotrans-
formation von Salicylverbindungen durch Zellkulturen der Weidenart Salix matsudana
turtuosa. PhD Thesis, University of Duesseldorf, Duesseldorf
Drewes FE, Staden JV (1995) Aspects of the extraction and purification of solasodine from
Solanum aculeastrum tissues. Phytochem Anal 6:203-206
Ehmke A, Eilert U (1993) Solanum dulcamara L. (Bittersweet): accumulation of steroidal alka-
loids in the plants and in different in vitro systems. In: Bajaj YPS (ed) Biotechnology in
agriculture and forestry, vol 21. Medicinal and aromatic plants IV. Springer, Berlin
Heidelberg, New York, pp 339-350
Frohne D (1993) Solanum dulcamara L - der i t t e r s i i ~ e Nachtschatten. Z Phytother 14:337-
342
Furuya T, Hirotani M, Kawaguchi K (1971) Biotransformation of progesteron and pregnenolone
by plant tissue cultures. Phytochemistry 10:1013-1016
Galanes IT, Webb DT, Rosario 0 (1984) Steroid production by callus and cell suspension cultures
of Solanum aviculare. J Nat Prod 47:373-376
412 G. Indrayanto et al.
Giulietti AM, Nigra HM, Caso 0 (1991) Solanum eleagnifolium Cav, (silverleaf night shade). In:
Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 15. Medicinal and aromatic
plants III. Springer, Berlin Heidelberg New York, pp 432-450
Groger D (1988) Terpenoid and steroid alkaloids. In: Constabel F, Vasil IK (eds) Cell culture and
somatic cell genetics of plants, vol 5. Academic Press, Orlando, pp 435-448
Grunwald C (1975) Plant sterols. Annu Rev Plant Physiol 26:209-236
Indrayanto G (1983) Steroide und Triterpene in Zellkulturen, Untersuchungen mit Zellkultu-
ren von Solanum laciniatum, S. wrightii und Costus speciosus. PhD Thesis, University of
Tuebingen, Tuebingen
Indrayanto G, Voelter W, Reinhard E (1983) Steroide und Triterpene in Zellkulturen. Chemiker
Z 107:238-239
Indrayanto G, Cholies N, Wahyudi (1985) Influence of fruit size of Solanum wrightii on its
solasodine content. Planta Med 51:470
Indrayanto G, Isnaeni, Sutarjadi (1986) Sterols in callus cultures of Solanum mammosum. Planta
Med 52:413
Indrayanto G, Rahayu R, Rahman A, Noerani PE (1993) Effect of calcium, strontium, and
magnesium on the formation of phytosteroids in callus cultures of Agave amaniensis. Planta
Med 59:97-98
Indrayanto G, Setiawan B, Cholies N (1994) Differential diosgenin accumulation in Costus
speciosus and its tissue cultures. Planta Med 60:483-484
Indrayanto G, Erawati T, Santosa MH (1995) Effect of I-arginine, casein hydrolysate, banana
powder and sucrose on growh and solasodine production in shoot cultures of Solanum
laciniatum. Plant Cell Tissue Organ Cult 43:237-240
Isfidiati (1988) Mikropropagasi Solanum mammosum L. Skripsi (BSc Thesis) Fakultas Farmasi
Universitas Airlangga, Surabaya.
Isnaeni (1986) Optimasi pembentukan kalus Solanum mammosum L dan identifikasi senyawa
steroidnya. MSc Thesis, Universitas Airlangga, Surabaya.
Karsana AGR (1988) Pengaruh penambahan Rhizopus arrhizus terhadap kadar sterol total dalam
kalus Solanum mammosum. Skripsi (BSc Thesis) Fakultas Farmasi Universitas Airlangga,
Surabaya
Kuc J (1984) Steroid glycoalkaloid and related compounds as potato quality factors. Am Potato J
61:123-140
Kupchan SM, Barboutis SJ, Know JR, Lau Cam CA (1965) Beta-solamarine tumor inhibitor
isolated from Solanum dulcamara. Science 150:1827-1828
Kusano G, Takahashi A, Sugiyama K, Nozoe S (1987a) Antifungal properties of Solanum alka-
loids. Chern Ph arm Bull 35:4862-4867
Kusano G, Takahashi A, Nozoe S, Sonoda Y, Sato Y (1987b) Solanum alkaloids as inhibitors of
enzymatic conversion of dihydrolanosterol into cholesterol. Chern Pharm Bull 35:4321-
4323
Macek TE (1989) Solanum aviculare Forst, Solanum laciniatum Ait. (Poroporo): in vitro culture
and the production of solasodine. In: Bajaj YPS (ed) Biotechnology in agriculture and foresty,
vol 7, Medicimal and aromatic plants II. Springer, Berlin Heidelberg New York, pp 444-
467
Miles OC, Wilkins A, Erasmus GL, Kellermann TS, Coetzer JAW (1994a) Photosensitivity in
South Africa VII. Chemical composition of biliary crystals from a sheep with experimentally
induced geeldikkop. Onderstepoort J Vet Res 61:215-222
Miles OC, Wilkins A, Erasmus GL, Kellermann TS (1994b) Photosensitivity in South Africa VIII.
Ovine metabolism of Tribulus terrestris saponins during experimentally induced geeldikkop.
Onderstepoort J Vet Res 61:351-359
Miller RH (1969) A morphological study of Solanum mammosum and its mammiform fruit. Bot
Gaz 130:230-237
Mizukami H, Terao T, Miura H, Ohashi 0 (1983) Glucosylation of salicyl alcohol in cultured plant
cells. Phytochemistry 22:679-680
Mufidah E (1988) Pengaruh ekstrak ragi terhadap kandungan sterol total dalam kalus Solanum
mammosum. Skripsi (BSc Thesis) Fakultas Farmasi Universitas Airlangga, Surabaya
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 5:473-497
Solanum mammosum L. (Terong Susu) 413
Nabeta K (1993) Solanum aculeatissimum Jacq: in vitro culture and the production of secondary
metabolites. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 24. Medicinal
and aromatic plants V. Springer, Berlin Heidelberg New York, pp 329-341
Paczkowski C, Wojciechowski ZA (1993) Glucosylation and galactosylation of diosgenin and
solasodine by soluble glycosyltransferas(s) from Solanum melongena. Phytochemistry 35:
1429-1434
Pranachita A (1992) Regenerasi kalus dari Protoplast Solanum mammosum dan S. wrightii. MSc
Thesis, Universitas Airlangga, Surabaya
Rahayu R (1988) Efek antifertilitas kalus Solanum mammosum pada mencit. MSc Thesis,
Universitas Airlangga, Surabaya
Reynolds EF (1993) Martindale. the extra pharmacopeia. 30th edn. The Pharmaceutical Press,
London, 994 pp
Ripperger H (1995) Steroidal alkaloids and sapogenins from roots of some Solanum species.
Plant a Med 61:292
Roddick JG, Rijneberg AL, Weissenberg M (1990) Membrane disrupting properties of the
steroidal glycoalkaloids solasonine and solamargine. Phytochemistry 29:1513-1518
Rusli H (1996) Pengaruh radiasi UV pada pertumbuahan dan kandungan kalus Agave amaniensis,
Solanum laciniatum (sl-7) dan Solanum mammosum (sm-1). Skripsi (BSc Thesis) Fakultas
Farmasi Universitas Airlangga, Surabaya
Samesti L (1988) Efek antifertilitas ekstrak aseton kalus dan buah Solanum mammosum pada
mencit. Skripsi (BSc Thesis) Fakultas Farmasi Universitas Airlangga, Surabaya
Santos ARD, Niero R, Filho VC, Yunes RA, Pizzolatti MG, Monache FD, Calixto JB (1995)
Antinociceptive properties of steroids isolated from Phyllanthus corcovadensis in mice. Planta
Med 61:329-332
Sarwetini KA (1988) Pengaruh penambahan buah pisang ambon memtah terhadap kultur kalus S.
mammosum dan S. wrightii. Skripsi (BSc Thesis) Fakultas Farmasi Universitas Airlangga.
Surabaya.
Sawariam I (1986) Studi kandungan buah Solanum mammosum L, Skripsi (BSc Thesis), Fakultas
Farmasi Universitas Airlangga, Surabaya
Sondakh R (1989) Biotransformasi progesteron dengan kultur kalus Solanum mammosum. MSc
Thesis, Universitas Airlangga, Surabaya
Stohs SJ, Rosenberg H (1975) Steroid and steroid metabolism in plant tissue cultures. Lloydia
38:181-192
Subroto MA, Doran PM (1994) Production of steroidal alkaolids by hairy roots of Solanum
avicculare and the effect of gibberelic acids. Plant Cell Tissue Organ Cult 38:93-102
Suharno (1986) Pcngaruh hormon pertumbuhan terhadap kecepatan pertumbuhan kultur
jaringan SoLanum mammosum dan S. tuberosum. Skripsi (BSc Thesis) Fakultas Farmasi
Universitas Airlangga. Surabaya
Susilowati AE (1987) Pengaruh konsentrasi sukrosa dalam media MS yang dimodifikasi terhadap
pertumbuhan kalus Solanum mammosum. Skripsi (BSc Thesis) Fakultas Farmasi Universitas
Airlangga. Surabaya
Syahrani A (1997) Biotransformasi derivat salisilat dengan kultur suspensi sel tanaman SoLanum
mammosum. PhD Thesis. Universitas Airlangga, Surabaya
Syahrani A, Indrayanto G, Sutarjadi. Wilkins A (1997) Bioconversion of salicylamide by cell
suspension cultures of Solanum mammosum. Chern Pharm Bull (in press)
Takechi M, Shimada S, Tanaka Y (1992) Structure-activity relationships of synthetic diosgenyl
diglycosides. Phytochemistry 31 :3280-3281
Tarigan P (1980) Sapogenin steroid. Penerbit Alumni, Bandung Indonesia, pp 96-103
Telek L (1977) Determination of solasodine in Solanum species. J Ph arm Sci 66:699-702
Telek L, Delphin H, Cabanillas E (1977) Solanum mammosum as a source of solasodine in the
lowland tropics. Econ Bot 31: 120-128
Thewles A, Parslow RA. Coleman R (1993) Effect of diosgenin on biliary cholesterol transport in
the rat. Biochem J 291:793-798
Thorne HV, Clarke GF, Suce R (1985) The activation of Herpes simplex virus by some
Solanaceae glycoalkaloids. Antiviral Res 5:333-343
Tsoulpha p. Doran PM (1991) Solasodine production from the self-immobilized Solanum
avicuLare cells. J Biotechno1 19:99-110
414 G. Indrayanto et al.: Solanum mammosum L. (Terong Susu)
Van Gelder WMJ (1989) Steroidal glycoalkaloids in Solanum species: consequences for potato
breeding and food safety. PhD Thesis, University of Wageningen, Wageningen
Van Steenis CGGJ, Den Hoed G, Eyma PJ (1951) Flora voor de Scholen in Indonesie. Noordhoff-
Kolf NV, Djakarta, pp 346-448
Vanek T, Macek T, Benes I, Navotny L (1985) Occurrence of betulinic acid in different callus
cultures of Solanum aviculare. Phytochemistry 24:3064-3065
Weiss en berg M, Levy A, Wasserman RH (1993) Solanum glaucophyllum Desf. (Duraznillo
Blanco): In vitro culture and the production of steroidal secondary metabolites. In: Bajaj YPS
(ed) Biotechnology in agriculture and forestry, vol 21. Medicinal and aromatic plants IV.
Springer, Berlin Heidelberg New York, pp 353-370
Wijono H (1987) Pengaruh sumber karbon terhadap pertumbuhan kultur jaringan Solanum
mammosum dan S. tuberosum. Skripsi (BSc Thesis) Fakultas Farmasi Universitas Airlangga,
Surabaya
Wu Y, Wu P (1991) Separation of C
25
epimers of steroidal sapogenins by reversed phase HPLC at
low temperature. Phytochem Anal 2:220-224