Solasodine

SOLASODINE

Gunawan Indrayanto, Achmad Syahrani, Robby Sondakh, and Mulja H. Santosa

Laboratory of Pharmaceutical Biotechnology Faculty of Pharmacy Airlangga University Surabaya, Indonesia

ANALYTlCAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

487

Copyright 0 1996 by Academic Press, lnc. All rights of reproduction in any form reserved.

388

GUNAWAN INDRAYANTO ET AL.

1. DESCRIPTION 1.1. Nomenclature 1.1.1. Chemical names 1.1.2. Synonym 1.2. Formula 1.2.1. Empirical 1.2.2. structural 1.2.3. CAS Registry No. 1.3. Molecular weight 1.4. Elementary composition 13.Appearance 1.6. Occurence 1.7. Use

2. PHYSICAL PROPERTIES 2.1. Melting point 2.2. Specific rotation 2.3. Solubility 2.4. Dissociation constant 2.5. Thermal Analysis 2.6. Spectm Properties of solasodine 2.6.1. Ultra Vioiet Spectrum 2.6.2. Absorbance Reflectance Spectrum 2.6.3. Near I n h Red Spectrum 2.6.4. Infra Red Spectrum 2.6.5. 'H-Nuc~EKMagnetic Resonance Spectrum 2.6.6. '3C-Nuclear Magnetic Resonance Spectrum 2.6.7. Mass Spectrum

3. ISOLATION OF SOLASODINE 4. BIOSYNTHESIS OF SOLASODINE

5. CHEMICAL CONVERSION OF SOLASODINE TO STEROID HORMONES

6. BIOTRAWFORMATION OF SOLASODINE

SOLASODINE

489

7. METHOD OF EXTRACTION AND QUANTITATNE ANALYSIS OF SOLASODINE

7.1. Method of extraction 7.2. Method of quantitative analysis 7.2.1. Titration 7.2.2. Colorimetric 7.2.3. Potentiometric 7.2.4. Gas chromatography 7.2.5. High Performance Liquid Chromatography 7.2.6. Radioimmunoassay 7.2.7. Thin Layer Chromatography - Densitometric 8. PHARMACOLOGICAL AND BIOLOGICAL ACTIVITIES OF SOLASODINE AND ITS GLYCOSIDES

490

GUNAWAN INDRAYANTO ET AL.

1. DESCRlPTION 1 . 1 . Nomenclature 1.1.1. Chemical names

Spirosol-5-en-3P-01; solasod-5en-3P-ol

1.1.2. Synonym Solancarpidine; Solanidhe-s; Purapuridine 1.2. Formula 1.2.1. Empirical C27H4,No,

1.2.2. smctural

1.2.3. CAS Registry No. 126-17-0 1.3. Molecular weight

413,6 1.4. Elementary composition C : 78.40 96;

H : 10.48 96; N : 3.39 96; 0 : 7.74 96

1.5. Appearance A fine white odorless crystalline powder; hexagonal plates crystal of solasodine can be recrystallized from methanol or by sublimation in high vacuum

SOLASODINE

49 1

1.6. Occurence Solasodine was accumulated in the species of Genus Solanum. Solasodine was produced commercially mostly from Solanwn laciniatwn, Solanum khasianwn and Solanwn marnmoswn (1,2,3). In Solanum plants, solasodine was found mostly as diglycosides, triglycosides and tetraglycosides (4). The sugar moiety of the important glycosides are shown in the table 1. Table 1. Sugar moiety of solasodine glycosides Glycosides J3+alamarginc

Rhamnose 01 (1-> 4) - Glucose p (1-> 3) - R

p-Solasonine Glucose p (1-> 3) Solamargine

Solasonine Solaradixine

I

Sugar moiety

- Galactose p (1->

>

Rhamnose a (1-> 2) Rhamnose a (1-> 4) Rhamnose a (1-> 2) Glucose

Glucose p (1-> 3)

>&lactose

p (1-> 3)

I

3) - R

-R

p (I-> 3) - R

Rharnnose a (1-> 2) >Galactose G~UCOS~ p (1-> 2 G I U C Op~(1-> 3)

p (1-> 3)-R

R = Solasodine

1.7. Use Solasodine is used as raw material for producing steroid hormone in pharmaceutical industry. 2. PHYSICAL PROPERTIES 2.1. Melting point 200 - 202O c (5) 2.2. Specific rotation [ aD ] at 25OC (c = 0.14 in methanol) = -98' ; [ aD ] =-113' (in chloroform) (5)

GUNAWAN INDRAYANTO ET AL.

391

2.3. Solubility The solubility data of solasodine are listed in table 2 Table 2. Solasodine solubility in various solvents

Benzene Methanol Ethanol 95 % Acetone n-Hexane Water Ether

Freely soluble. Freely soluble.

9.5 5.0 3.5 <1.0 <1.0 Practically insoluble'

* Data from Merck Index (5) 2.4. Dissociation constant pK, in 60 % ethanol was 6.31 (6). 2.5. Thermal Analysis The DSC (differential scanning calorimetry) thermogram of solasodine was obtained on a Shimadzu DT-30 Thermal Analyzer. The instrument was calibrated with indium standard. The heating rate was 10°C/min. The thermogram is presented in figure 1. The DSC curve revealed an endothermic residual moisture peak on a single sharp endothermic melting peak (200OC) of solasodine. 2.6. Spectral Properties of solasodine 2.6.1. Ultra Violet Spectrum The U V (ultra violet) spectrum of solasodine in methanol (ca. 40 ppm) was obtained on a Hewlett Packard 8452A photo diode array spectrophotometer. It exhibits a maximum at 206 nm. The UV spectrum and its first order derivative spectrum are presented in figure 2A and 2B. 2.6.2. Absorbance Reflectance Spectrum The visible absorbance reflectance spectrum of solasodine spotted on Kieselgel GF254 precoated plate (EMerck), eluted with chloroform:

Figure 1. DSC thermogram of solasodine (Sigma).

GUNAWAN INDRAYANTO ET AL.

194

I

0.72649,

i

I A I

A

%z 0.43589-

2c:

20.29059m

<

I

0.14530/

I 200

\

nm,

2.50

300

350

400

B 0.06829-

-w 0.03927 >

pO.OIa26 U

Figure 2. UV spectrum of solasodine (Sigma)in methanol (A) and its fxst order derivative spectrum (B).

2.000

1.600

* sr

1.200

8

0.800

3 0.400

81

O.Oo0

- 0.200 -

I

400

,

.

. . , . 450

.

.

.

I

500

.

,

I

.

I

550

.

.

.

-

1

600

.

I

-. 650

Figure 3. Visible absorbance ref'lectantce spectrum of solasodine (Sigma) on Kieselgel GF254 precoated plate (Merck). BL: base line.

K1 nm

GUNAWAN INDRAYANTO ET AL.

496

methano1:diethyl amine (20:2:0.5) and Visualized by anise aldehyde-sulphuric acid (7)spray reagent (10OoC,10 minutes), is shown in figure 3. It shows a maximum at 385 nm. The spectrum was recorded on a Shimadzu CS-930TLC Scanner. 2.6.3. Near Infra Red Spectrum The NIR (near infra red spectrum) of 0.5 % solasodine in CCl, was recorded by using a Shimadzu 365 spectrophotometer. The N I R spectrum of solasodine is shown in figure 4. The Xmx data are presented in table 3. Table 3. NIR characteristics of solasodine Amax (nm)

Assignment

1413

OH stretching

1527

NH stretching

2309

CH stretching

2352

2.6.4. Infra Red Spectrum The IR (infra red) spectrum of solasodine as KBr disc (1.5mg/ 100 mg) was recorded on a Jasco 5300 FT IR spectrophotometer. The IR spectrum of solasodine is shown in figure 5. The characteristic bands of solasodine are given in the table 4. Table 4. IR characteristic bands of solasodine ~~

~

Frequencies (crn-') 3455 3362 2843,2967 1690 1675 1451, 1344 1379 1060, 1239

I

Assignment Stretching vibration of OH Stretching vibration of NH Stretching vibration of CH steroid skeleton Stretching vibration of C = C bond Deformation vibration of NH Deformation vibration of methyl Deformation vibration of methylene Stretching vibration of C-QC

9"

0

c 5 ABSORBANCE

'0 Q

0

F!

3

-

100.00

%T

0.001.. 4600.0

1

.

4000.0

.

.

.

I

3000.0

.

.

. .

I

2000.0

I

1ooo.o

Figure 5. IR spectrum of solasodine (Sigma) in KBr disc.

400.0

(cm-')

SOLASODINE

499

2.6.5. 'H-Nuclear Magnetic Resonance Spectrum The 'H-NMR spectrum of solasodine (Figure 6A and 6B) were

determined in CDCl, at 90 MHz employing a Hitachi R-1900 FT NMR using TMS as internal standard. The chemical shift values are presented in Table 5. Table 5. 'H-NMR chemical shifts of solasodine Proton assignment Me-18 Me-19 Me-2 1 Me-27 H-6 H-3a H-16 H-26a, p H ~ u P,

Chemical shift (6 ppm) Pyridine - D5

CDCI,

0.82 (s) 1.03 (s) 0.98 (s) 0.88 (s) 5.32 (br.d) 3.50 (m) 4.27 (4;6.5,6.5, 6.5 I+) 2.57-2.65 (m; not resolved) 2.21-2.30 (m;not resolved)

1.05 (s) 0.91 (s) 1.13 (s) 0.84 (s)

3.80 (m) 4.43 (4;6.7, 6.7, 6.7 Hz)

By using two-dimensional NMR technique (COSY,one bond, long range 'H-I3C chemical shift correlation and NOESY),obtained on a Bruker

AMX-500 NMR spectrometer (500MHz), Puri et al (8) could elucidated all the proton chemical shifts of solasodine. The data are presented in the table 6.

5x

....

-.. 5.00

. . , , 4.00

..,. ._.. 3.00

' . . . ' I . . . ' -

2.00

Figure 6A. Proton NMR spectruni of solasodine (Sigma) in CDCh

'.OO

PPM

501

CUNAWAN INDRAYANTO ET AL.

502

Table 6. 'H-NMR chemical shifts of solasodine (6 ppm, CDCl,)* _e_l

Proton assignment -

l-a

1-P 22-9

~.

3 4-a 4-P 6 7* 7-P 8 9 11 12-a 12-P 14

Chemical shift

1.oo 1.80 1.78 1.40 3.42 2.24 2.16 5.23 1.48 1.97 1.58 0.90 1.48; 1.41+ 1.10 1.68 1.02

1 Proton assignment 151543 16 17 Me-18 Me-19 20 Me-2 1 23 24-

24-P 25 2626-P Me-27

Chemical shift 1.95 1.25 4.20 1.65 0.74 0.95 1.81 0.88 1.56 1.55 1.35 1.48 2.58 2.52 0.77

+) not resolved

*)

Data from Puri et al. (8)

2.6.6. '3C-Nuclear Magnetic Resonance Spectrum The broad band decoupling 13C-NMR spectrum (Figure 7) and the J modulation spin echo 13C-NMR spectrum (Figure 3) with 1/J = 7 p sec of solasodine in CDCl were recorded on a Hitachi R-1900 FT NMR (22.6MHz) using TMZ as internal standard. The spectral assignments are presented in Table 7. The data are identical with previously reported spectrum (9, 10).

TMS

J! L L

Figure 7. Broad band decoupled 13C-NMR spectrum of solasodine (Sigma) in CDC13.

PPM

J

CH,CH3

C,CHz

1

TMS

CDCh

50.00

Figure 8. J modulation spin echo (1/J = 7 ItSec) I3C-NMR spectrum of solasodine (Sigma) in CDC13.

00.00

PPM

505

SOLASODINE

Table 7. 13C-NMR chemical shifts of solasodine Carbon No.

1

-~

Chemical shifts

Chemical shifts

Carbon No.

(6 P P d

(6 P P d

1

37.3

15

32.1

2

31.6

16

78.7

3

71.6

17

62.8

4

42.3

5

140.7

19

19.4

6

121.3

20

41.3

7

32.1

21

15.3

8

31.4

22

98.1

50.1

23

34.1

10

36.7

24

30.3

11

20.9

25

31.4

12

39.9

26

47.7

13

40.5

27

19.3

14

56.5

9 ~~

I

18

I

16.4

~~

2.6.7. Mass Spectrum The MS (mass spectrum) of solasodine presented in Figure 9 and Figure 10. were obtained by electron impact (EI) and chemical ionization (CI) using methane as reagent on a Jeol JMS-DX-303 Mass Spectrometer. The ionizing electron beam energy was at 70 eV. The main fragments of solasodine are given in the table 8.

GUNAWAN INDRAYANTO ET AL.

506

Table 8. The main fragments of solasodine Species

El

CI

M+ + 29

442 (8)

M+ + 1

414 (55)

M+ + 1 - 18(H,O)

396 (58) 413 (27)

M+ W

-

15(CH,)

398 (3)

M + - 28(C2H,)

385 (13)

C,H2,NO+

271 (2)

a*

138 (70)

138 (10)

b’

114 (100)

114 (27)

* see figure 1 1 The fragments of m/z 138 and 114 are characteristic for spirosolane ring of solasodine (11, 12). The EI spectrum of solasodine is identical with previously published spectrum (13).

MIIZ

Figure 9. EI-MSspectrum of solasodine (Sigma)

RELATIVE ABUNDANCE

SOLASODINE

509

+

4.

O

mlz 138

?

3

m/z 114

Figure 11. Characteristic fragmentation of solasodine.

510

GUNAWAN INDRAYANTO ET AL.

3. ISOLATION OF SOLASODINE In our laboratory, solasodine was isolated from fruits of SoZunum wrightii with the following method, as reported by Indrayanto et al.(14).

Fresh ripe fruits (3 kg) were cut into small pieces and homogenized in a blender with 4 times methanol-acetic acid 3 % until slurry was formed. The slurry was refluxed for 30 minutes then filtered. Each 50 ml of filtrate was treated with 5 ml HC1 conc. and hydrolyzed by refluxing for 3 hours on boiling water bath. After neutralized with 10 %. NaOH, the alkaloids was extracted with chloroform. The chloroform extract was evaporated in vacuo to yield a dark brown semisolid mass (22 g),then purified through a column of A1 0, with chloroform as the eluting solvent. After evaporating the cgloroform, the residue was chromatographed on silica gel 60 with benzene:acetone (15: 1) as eluting solvent. Recrystallization with methanol gave 3.63 g pure solasodine (TLC, UV,TR, MS). 4. BIOSYNTHESIS OF SOLASODINE

The biosynthetic pathway of solasodine followed the general pathway of steroid biosynthesis, starting from acetylcoenzym A via the in termediates mevalonic acid, squalene, cycloartenol and cholesterol. The nitrogen atom was introduced through simple replacement of the terminal hydroxy group by an amino group (15). In solanidine the donor molecule was an amino acid arginine (16). The main steps of the pathway (partially hypothetical) of cholesterol to solasodine in are presented in figure 12 (15,17,18). The figure shows that the formation of ring E can precede or after the formation of ring F.

5. CHEMICAL CONVERSION OF SOLASODINE TO STEROID HORMONES In order to obtain steroid hormone like materials, ring E and F of solasodine attached to C16 and C 17 first must be removed. Solasodine can be degraded into 16-dehydropregnenoloneacetate (16-DPA), which is an excellent starting material for the preparation of most type of steroid hormone (Marker degradation). In figure 13 the conversion of solasodine to some steroid hormone is presented (19,20).

SOLASODINE

511

Fmesyl pyrophosphate

Mevalonic acid

Acetyl-CoA

HO

#-@ Squalcne

Cycloartenol

Dormantino1

Dormanunonc

4 22, 26-Epimino-Ssholesten-3n,16R-diol

no

26-Am:no-160-hydroxycholer~rol

1

Verazine I

HO

-

26-A iiiiiiodihydrodiosgenin

Etiulinr

1

\ & ."-OH n " ^ * ' .NO &\ % Solasodine I40

Teinemine

Figure 12. The biosynthetic pathway of solasodine

512

GUNAWAN INDRAYANTO ET AL

J

Figure 13. Chemical conversion of solasodifie to steroid hormone.

SOLASODINE

513

6. BIOTRANSFORMATION OF SOLASODINE

Patel et al. (10) reported recently that the fungus Cunninghumella ekguns could transform solasodine into solasod-5ene- 38, 7BIB-di01, solasod-5ene-38,7adioland 38- hydroxysolasod-5-en-7ae. In contrast, incubation of solasodine with fungus PenicilZiumpanrlum gave solasod4ene-3a1e and the 6-methylsalicylicacid salt of solasodine. By using Mycobucteriumphlei solasodine could be transformed to 4-androstene-3,17dioneand 1,4-androstadiene-3,17dione(21). Shulz et al. (22) reported that in leaves extract of Solmum luciniutum, solasodine converted to its glucoside by a solasodine glucosyltranferase. 7. METHOD OF EXTRACTION AND QUANTITATIVE ANALYSIS OF SOLASODINE 7.1. Method of extraction For the extraction of solasodine as glycoalkaloid, the plant or cell culture materials could be extracted with methanol ( 23), ethanol 95 % (24,25), methano1:chloroform (2: 1) (26), 3 - 5 % aqueous acetic acid (27,28), 5 % acetic acid in methanol (29), 2 % acetic acid in 90 % ethanol (30) or 2 % oxalic acid (31). To determine the aglycone (solasodine) the extracts must be hydrolyzed by 1N HC1, neutralized with NaOH or ammonia, then extracted with organic solvent (chloroform). To determine solasodine, the biomass also could be directly hydrolyzed with 1N HCl (32,33) or 2N HCl in methanol (34). For separating non polar components (eg. free sterols) , the biomass was extracted with chloroform before hydrolized (35). To minimize the decomposition of solasodine into solasodiene, Segal et al. ( 36) recommended using of non aqueous low boiling point alcohols and acid concentration not exceeding 1N. By using two phase system consisting carbon tetrachloride and HCl as hydrolized mixture, Van Gelder (17) could prevent the degradation of the steroid alkaloid. Oven drying above 100°C of the plant materials also lead to solasodine loss (37). 7.2. Method of quantitative analysis 7.2.1 Titration Method of Eldridge and Hockridge (26)

51.2

GUNAWAN INDRAYANTO ET AL.

To determine the glycoalkaloid content in Solanum prycamhum, the dried berries was extracted with methanol :chloroform (2: l), then the extract was mixed with 0.8 % Na,SO,, shaken in a separatory funnel and ailowed to settle overnight. The methanol phase was collected, dried, and dissolved in 2N H SO,. This solution was heated for 2 hours and made basic with 4fi NaOH. The aglycone was extracted 3 times with benzene. After evaporating the residue was dissolved in methanol and titrated with a solution of 0.067 % bromophenol blue and 10 % phenol in absolute methanol, against a blank of methanol. Method of Valovich (31) After extraction of the glycoalkaloid with 2’% oxalic acid, hydrolyzed with HQ, the hydrolysate was added a 50 % solution of caustic soda to bring the pH to 9.5 for precipitating solasodine. The solasodine was extracted with neutral chloroform, and the chloroform solution was titrated with O.OO5N solution of n-toluene-sulphonic acid in chloroform, with 0.1 % solution of dimethyl yellow as indicator. 7.2.2. Colorimetric Method of Birner (25) Finely powdered materials was refluxed with 95 % ethanol for 30 minutes, filtered, and the ethanol extract was collected. After evaporating, the residue was hydrolyzed with 1N HCI,then neutralized with 1N NaOH. The aglycone was comphed with methyl orange and the colored complex extracted into chlorohrm and determined colonmetrically at 430 nm. Method of Khdagi et al. (24) In this method, solasodine was dissolved in 95 % ethanol, after addition of phosphate buffer pH 7.5, a solution of 0.1 % bromothymol blue in alcohol 50 %! was added to the mixture. The bromothymol blue-solasodine complex was extracted using benzene, and measured on spectrophotometer at 400 nm before 45 minutes. Method of Nigra et al. (38). Firstly the hydrolyzed sample was alkalinized with 60 % NaOH then solasodine was extracted with chloroform. After addition of 2. 1i4M of brornothymol blue in borax buffer pH 6.8 and mixed in a vortex-mixer,

SOLASODINE

515

the aqueous phase was taken out, added solution of methanolic 0.01N NaOH to the chloroform phase, then measured at 610 nm. 7.2.3. Potentiometric Method of Telek (27) The steroid glycoalkaloids were extracted from freshly harvested fruits with 2 % acetic acid and methanol. After hydrolysis, the common aglycone solasodine was extracted in benzene. An aliquot was mixed with an equal volume of acetone and titrated potentiometrically with 0.005 N perchloric acid in dioxane, using glass and silver electrodes for determination. 7.2.4. Gas chromatography

Methof of Carle (34) In this method, the freeze dried biomass was hydrolyzed using 2N HCl in methanol. After neutralization with ammonia 25 % , solasodine was extracted by chloroform. An aliquot of the chloroform phase was injected to a GC with the following condition, column glass (1/8 inch x 6 ft) packed with 3 % SE-30 (Gas Chrom Q, 100-120 mesh); FID and injector temperature were 30OoC; column temperature was isothermal at 250OC. By this condition solasodine and diosgenin/tigogenin were separated and quantitatively determined from Solmum plant materials. Method of Indrayanto (39) By using a glass column ( 2m x 2mm id.) packed with 3 % OV-1 on Gas Chrom Q ( 100-120 mesh); FID and injector temperature 30OoC; oven temperature was programmed from 200 to 280°C, S°C/minute, solasodine, hecogenin, diosgenin and sterols (cholesterol, campesterol, stigmasterol, sitosterol and isofucosterol) could be good separated. Method of Van Gelder (17) To prevent steroid alkaloid, degradation, van Gelder was used two phase system of carbon tetrachloride and HC1 to hydrolyze the plant materials. For analyzing the steroid alkaloids two systems of GC were used by the author. System 1 : glass column (lm x 2mm id.) packed with 10 % SE-30 on Chromosorb W HP (80-100 mesh); maximum operating temperature were 325OC (isothermal), 35OoC (programmed); FID temperature 350OC;

516

GUNAWAN INDRAYANTO ET AL.

injector temperature 325OC. System 2 : fused capillary column of 50m x 0.22 mm id., coated with CP-Sil 5 (film thickness 0.12 pm or CP-Sil 19 (film thickness 0.19 pm); oven temperature were 30O0C (isothermal), 325OC (programmed); R D and injector temperature were as the same as system 1. With both systems, solasodine, solanidine, demissidine, tomatidine, solasodiene and solanthrene could be separated. By using two detectors (FID and NPD), the identity of 21 steroid alkaloids can be elucidated by retention indexes and NPD/FID response ratio.

7.2.5. High Performance Liquid Chromatography Some of HPLC methods in the determination of solasodine or its glycosides that have been published are listed in table 9. By using combination of HPLC and thermospray mass spectrometry (LC-MS), the structure of the steroid alkaloid glycosides could be elucidated. This method seems well suited for a screening of a complex mixtures for a certain steroidal alkaloids (40). 7.2.6. Radioimm unoassay Weiler et al. (46) have reported a rapid, spesific and sensitive RIA for the determination of solasodine in vegetative and generative parts of Soiunwn luciniatwn. The antiserum added did not react at all with sterols, solanidanes and spirostanols, but showed strong reactivity with tomatidine and solasodine glycoalkaloids. The assay allowed the detection as little as 0.7 ng solasodine glycosides. 7.2.7. Thin Layer Chromatography - Densitometric Some of the published methods of Thin layer chromatography (TLC) of solasodine are listed in table 10. In our laboratory the solasodine content of in the vitro cultures of S o l a w spp. was determined by densitometric method. Firstly the dried biomass was extracted with chloroform 3 times on a vortex mixer to remove the non polar components (eg. free sterols), then hydrolised with 2N HCl in methanol (2 hours, 75'C), After neutralised with 10N NaOH, solasodine was taken out by chloroform. The chloroform extract was spotted on precoated Kieselgel G 60 F254 (Merck), and chloroform : methanol : diethylamine (20:2:0.5) was used as eluent. The solasodine

M

sifidcacidradal

w

I acebnibae:wster(77.5:22.5);pH4.0

a

sample

w 240,

Kasslbrataetrl. (21)

205nm

-,17 Qne, 1,4 adrostadecle3,17dare,~@EO&IO

W205nm

sdssodne,-ne

Vogd ad.(23)

WMom

acet#libile :Pic eS :TEA (83:17 :0.1); pH 2.8

W205Nll

:0.1 MThkmer(9: 1)

w 210 nm IWWI6 Hurler(41) W 213 nm

pBondapakC18

I metranol:O.OlMTriskner(75:25)

Hurleretal.(42)

WMSn

a-,B-,wlvcosidea daaodm

Table 9. HPLC methods for determining solasodine and its glycosides

I nHaxas:EW

(1:l) (1:l) C W : M H (23:2) clt2az:MsoH (9:l) ctt2c12:AceQne (4: 1) nHaxas :h b l e (1 : 1) CHzClz :&OH :AmUc acid(= : 13: 2) nHaxas:EtOH

PqNoj

Me0H:CHQ (1:9)

Table 10. TLC methods for Analysing of solasodine

SOLASODINE

519

spots was detected by anise aldehyde-H2S04reagent (lOO°C, 5-10 minutes). Quantitation was done by measurmg at the maximum absorbance reflectance (385 nm). The determination of solasodine was made by calculation with a calibration graph obtained using solasodine (Sigma) as external standard on the same TLC plate. With this method linearity of solasodine was achieved from 0.4 to 1.6 pg/spot (r=0.998, n=7, V =2.1 %); LOD (limit of detection)= 0.11 pg/sjmt; LOQ (limit of q1,?&titation)=0.31 pg/spot; 'mean accuracy @ f SD) with standar addition method was 98.92 f 2.35 %, n = 14; RSD of precision determination was 1.87 % (n=10). 8. PHARMACOLOGICAL AND BIOLOGICAL ACTMTIES OF SOLASODINE AND ITS GLYCOSIDES As early as 1965 Kupchan et al. (53) demonstrated a tumorinhibiting activity of the glycoside 13 solamargine. Cham et al. (54) showed t!!at glycoalkaloid solasonine, solamargine and another mixture of glycosides containing aglycone solasodine, had on antineoplastic activity against Sarcoma 180 in mice. Solasodine inhibited the growth some fungal strains, although its activity was less compared to solafloridine and verazine (55). Some steroidal alkaloids including solasodine showed inhibitory effect on the enzymatic conversion of dihydrolanosterol into cholesterol (56). The steroidal glycoalkaloid solasonine and solamargine showed a membrane-disrupting properties of phosphatydylcholinekholesterol liposomes at concentration > 50 pM (57). A slight acetylcholinesteraseinhibitory activity of solasonine and solamargine was also reported (58).

Recently Frohne (59) reported that solasodine had a cortisone like effect such as anti phlogistic or reduction of blood vessel permeability. Solasodine also can prevent of anaphylactic shock in guinea pig. In a clinical trial a dose of 1 mg solasodine citrate which was given 2 times a day showed cardiotonic effect. This clinical trial also showed that solasodine gave a desensitization effect especially in patient with a rheumatoid polyarthritis.

520

GUNAWAN INDRAYANTO ET AL.

References 1. Telek,L., Delphin,H., Cabanilias,E., (1977) Economic Botany 3 1: 120-128. 2. Nigra,H.M., Alvarez,M.A., Giulietti,A.M., (1990) Plant Cell Tissue and Organ Cultures 21: 55-60. 3. Sudiarto, Chairani,F., Rosita, S.M., Wahid,P. (1985) Jurnal Litbang Pertanian 4: 71-76. 4. Macek, T.E., (1989) Solanum aviculare Forst., Solanum laciniatum Ait. (poroporo): In vitro cultures and the production of solasodine, in :Bajaj,Y.P.S. (ed.)Biotechnology in Agriculture and Forestry Vol. 7, Springer Verlag, Berlin Heidelberg, pp.444-467. 5. The Merck Index, 11thEd.(1989),Merck & CO., Inc, p. 520. 6. Bentley, K.W. and Kirby, G.W. (1972) Elucidation of Organic structure by physical and chemical methods, 2nd ed., Interscience, p. 565. 7. Europaiesches Arzneibuch,Band I, I1 (1976), Wissenschaftliche Verlagsgesselschaft MBH Stuttgart Goviverlag GMBH, Frankfurt. 8. Puri, R., Wong, T.C., Puri, R.K. (1993) Magnetic Resonance in Chemistry 31:278-282. 9. Radeglia, R., Adam, G . , Ripperger, H. (1977)'Tetrahedron Letter 11:903-906. 10. Patel, A.V., Blunden, G., Crabb, T.,4. (1994) Phytochemistry 35: 125-133. 11. Budzikiewicz, H., Wilson, J.M.. Djerassi, C. (1962) Monatshefte fiir Chemie 93: 1033-1041. 12. Budzikiewicz, H., Djerassi, C., William, D.H., (1964) Structure elucidation of natural products by Mass Spectrometri, Vol 11, Holden Day Inc., San Fransisco, hndon, Amsterdam, pp. 110-120. 13. Budzikiewicz, H. (1964) Tetrahedron 20: 2276. 14. Indrayanto, G., Tutuk Budiarti, Soebahagiono, Emma, Sutarjadi (1979) The solasodine content of Solanm grandiflonun, paper presented in UNESCO Regional Seminar on Medicinal Plants Bangkok, Thailand. 15. Heftmann, E. (1983) Phytochemistry 22: 1843-1860. 16. Kaneko, K., Tanaka, M.W., Mitsuhashi (1976) Phytochemistry 15: 1391. 17. Van Gelder, W.M.J.(1989), PhD thesis, Agriculture University Wageningen

SOLASODINE

52 1

18. Tschesche, R. and Brennecke, H.R. (1980) Phytochemistry 19: 1449-1451. 19. Wall, M.E. (1986) Status of raw materials for production of oral contraseptives in Indonesia, Paper presented in the Workshop of Biotechnology of Steroid Compounds as Contraceptives and drugs, Jakarta, Indonesia. 20. Sebek ,O.K. ,(1986) Production of steroid compound by fermentation, paper presented in the Workshop of Biotechnology of Steroid Compounds, Jakarta, Indonesia. 21. Kartasubrata,J., Loyniwati, Jarnilah, Karossi, A.T. (1993) Indonesian Journal of Applied Chemistry 3: 61-68. 22. Schulz,D., Eilert,U., Ehmke, A. (1993) Planta Media Supplement 59: A649. 23. Vogel,H., Jatisatienr, A., Bauer, R. (1990) Angew. Botanik 64: 393-400. 24. Khafagi, S.M., Amin, S.W., Hassanin, R. (1970) Planta Medica 21: 139-141. 25. Birner, J. (1969) Journal of Pharmaceutical Sciences 58: 259. 26. Eldridge, A.C., and Hockridge, M.E. (1983) Journal Agric. Food Chemistry 31: 1218-1220. 27. Telek, L. (1977) Journal of Pharmaceutical Sciences 66: 699-702 28. Cham, B.E. and Wilson, L. (1987) Planta Medica 59-61. 29. Hunter, I.R., Heftmann, E. (1983) Journal of Liquid Chromatography 6 ~281-289. 30. Chowdhury, A.R., Prasas, R.N., Uddin. A. (1979) Quart. Journal of Crude Drug Research 17 : 137-138. 31. Valovich, N.A. (1965) Meditzinskaya Promyshlennost USSR 19:

4548. 32. Ehmke, A. and Eilert, U. (1993) Sulunwn dulcumuru L.: Accumulation of steroidal alkaloids in the Plants and in different in vitro cultures, in : Bajaj, Y.P.S. (ed.) Biotechnology and Forestry, Vol. 21, Medicinal and Aromatic Plants VI, Springer Verlag, Berlin Heidelberg, pp.339-349. 33. Chandler, S., Dodds, J. (1983) Plant Cell Reports 2: 69-72. 34. Carle, R. (1979) Ph D thesis, University of Tuebingen. 35. Indrayanto, G., Studiawan, H., Noor Cholies (1994) Phytochemical Analysis 5 : 24-26. 36. Segal, R., Breuer, A., Milo-Goldzweig, I. (1978) Journal of Pharmaceutical Sciences 67: 1169-1170.

512

GUNAWAN INDRAYANTO ET AL.

37. Crabe, P.G., Fryer, C., (1982) Journal of Pharmaceutical Sciences 71: 1356-1362. 38. Nigra, H.M., Caso, O.H., Giulietti, A.M., (1987) Plant Cell Reports 6: 135-137. 39. Indrayanto, G., (1983) Ph D thesis, University of Tuebingen. 40. Ehmke. A., Schiebel, H.M., McDowell, M. (1987) Pharmaceutisch Weekblad Scientific Edition 9: 232. 41. Heftmann, E., Hunter, I. R. (1979) Journal of Chromatography 165: 283-299. 42. Hunter, I.R., Walden, M.K., Heftmann, E. (1980) Journal of Chromatography 198: 363-366. 43. Crabbe, P.G. and Fryer, C. (1980) Journal of Chromatography 187: 87- 100. 44. Indrayanto, G, Utami, W., Santosa, M.H., Syahrani, A., Diosgenin, Analytical . Profile of Drug Substances and in: Brittain, H.G.(4) Excipients Vol. 23., Academic Press, in Press. 45. Ehmke, A. and Eilert, U. (1986) Plant Cell Reports 5: 31-34. 46. Weiler. E.W., Kriiger, H., Zenk, M.H. (1980) Planta Medica 39: 112-124. 47. Hunter, I.R., Walden, M.K., Wagner, J.R., Heftmann, E. (1976) Journal of Chromatography 118 :259-262. 48. Puri, R.K. and Bhatnagar, J.K. (1975) Phytochemistry 14: 2096. 49. Willuhn, G. (1966) Planta Medica 14: 408-419. 50. Willuhn, G. and Kun-anake, A. (1970) Planta Medica 18:354-359 51. Kadkade, P.G. and Rolz, C. (1979, Phytochemistry 16:1128 52. Kartasubrata, J., Fitri, T.Y., Halomoan, V.A., Lotulung, P., Buchori, Karossi, A.T., (1993), Annales Bogorienses 2: 12-15 53. Kupchan, S.M., Barboitis, S.J., Know, J.R., Lau Cam, C.A. (1965) Science 150: 1827-1828. 54. Cham, B.E., Giliver. M., Wilson, L. (1987) Planta Medica 34-36. 55. Kusano, G., Takahashi. A . , Sugiyama, K., Nozoe, S . (1987) Chemical Pharmaceutical Bulletin 35: 4862-4867. 56. Kusano, G., Takahashi, A., Nozoe, S., Sonoda, Y.,Sato, Y. (1987) Cheinical Pharmaceutical Bulletin 35: 4321-4323. 57. Roddick, J.G., Rijnenberg. A.L., Weisenberg, M. (1990) Phytochemistry 29: 1513-1518. 58. Roddick, J.G. (1989) Phytocheinistry 28: 2631-2634. 59. Frohne, D. (1993) Zeitschrift fur Phytotherapia 14: 337-342.