Sterol and Triterpene Profiles of the Callus Culture of Solanum mammosum

This study aimed to compare the sterol and triterpene profiles of two types of Solanum mammosum callus cultures, i.e., compact globular structure (CGS) and normal fine (F) calluses. The CGS callus resulted from the differentiation of the F callus culture after many years of subculturing. The growth rate, microscopic characteristics, and morphologies of the two callus types were determined and compared. Sterols and triterpenes were identified through thin-layer chromatography, gas chromatography–flame ionization detection, and gas chromatography–mass spectrometry analyses. The growth rate of the CGS callus was lower than that of the F callus. Microscopic identification revealed that thick, lignin-containing cell walls formed in the CGS callus but not in the F callus. The chromatographic analysis suggested that the CGS and F callus cultures had different sterol and triterpenoid profiles. The sterols and triterpenes produced by the CGC culture were more diverse than those produced by the F callus culture.


Introduction
Sterols can be found in all eukaryotic organisms as membrane components that regulate the fluidity and the permeability of phospholipid bilayers. Most plant cells can produce sterols, such as cholesterol, campesterol, and β sitosterol, via the cycloartenol pathway [1]. Sterols in plants originate from cycloartenol, whereas those in fungi or animals are derived from lanosterol [2]. Sterols are members of the terpenoid family. Specifically, sterols are triterpenoids. Terpenoids are produced by plants and have various basic functions in growth and development but mainly participate in chemical interactions and protection against abiotic and biotic environmental stressors [3]. Several sterols have important biological activities, including anti-inflammatory, antidiabetic, anticancer, and lipid-lowering activities [4]. Triterpenes, such as betullinic acid, are natural products that exert activities against a variety of cancer types by directly influencing mitochondrial membrane permeabilization [5].
Sterols can be found in all eukaryotic organisms as membrane components that regulate the fluidity and the permeability of phospholipid bilayers. Most plant cells can produce sterols, such as cholesterol, campesterol, and βpathway [1]. Sterols in plants originate from cycloartenol, whereas those in fungi or animals are derived from lanosterol [2]. Sterols are members of the terpenoid family. Specifically, sterols are triterpenoids. Terpenoids are produced by plants and have various basic functions in growth and development but mainly participate in chemical interactions and protection against abiotic and biotic environmental stressors [3]. Several sterols have important biological activities, idiabetic, anticancer, and lowering activities [4]. Triterpenes, such as betullinic acid, are natural products that exert activities against a variety of cancer types by directly influencing mitochondrial membrane permeabilization [5]. and triterpenes have been identified in several spp. Cholesterol, stigmasterol, sitosterol, isofucosterol, lanosterols, lupeol, betulin, betulin aldehyde, and betulinic acid have been identified [6,7]. Cholesterol, sitosterol, 28-isofucosterol, methylene cycloartanol have also been found in (current name: S. glaucophyllum) [8]. The callus cultures of and S. wrightii produce cholesterol, campesterol, stigmasterol, and β-sitosterol but not solasodine [7,9]. The application of cell cultures of production of phytosteroids and the biotransformation of some chemical compounds have been reviewed [10].

Solanum mammosum
Plant Biotechnology Research Group, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Solanum mammosum callus cultures, i.e., compact globular structure (CGS) and normal fine (F) calluses. The CGS callus resulted from the differentiation of the fter many years of subculturing. The growth rate, microscopic characteristics, and morphologies of the two callus types were determined and compared. Sterols and triterpenes were identified through thin-layer chromatogmass spectrometry analyses. The growth rate of the CGS callus was lower than that of the F callus. Microscopic identification revealed that thick, lignincallus. The chromatographic analysis suggested that the CGS and F callus cultures had different sterol and triterpenoid profiles. The sterols and triterpenes produced by the ) [8]. The callus cultures of S. mammosum produce cholesterol, campesterol, sitosterol but not solasodine [7,9]. The application of cell cultures of S. mammosum for the and the biotransformation of some chemical compounds have been reviewed [10]. Some plant cell cultures can self-develop compact globular structure (CGSs). In general, the secondary metabolite profiles of CGS cultures are different from those of normal callus cultures [11][12][13]. In addition, CGS cultures exhibit cell wall thickening. Two forms of S. callus cultures, CGS and normal (F), are available in our laboratory ( Figure 1

Materials and Methods
Preparation of callus cultures. The CGS and F callus cultures of S. mammosum were grown in a 300 mL Erlenmeyer flask containing 50 mL of modified Murashige-Skoog medium supplemented with 7 g/L agar, 30 g/L sucrose, 2 mg/L kinetin, and 0.5 mg/L 1-Naphtaleneacetic acid at 25°C ± 2°C under continuous light (ca. 2000 lux). The cultures were subcultured every 4 weeks of incubation as described in our previous work (Indrayanto et al., 1998). Growth rate was determined by measuring the weight of four callus cultures every week by calculating the ratio of the callus weight at certain time (n th week) to the initial inoculation weight (n = 0). Data were reported as average rate ± standard deviation (SD). Sample preparation. Extraction was performed in accordance with a previously published method [7]. Oven-dried (40°C; moisture content 10%) powdered callus (6 g) was extracted with 50 mL of n-hexane by ultrasonification (3 × 15 min). The extract and residue were separated through filtration. The residue was further extracted with acetone (50 mL) using the same procedure. All collected extracts were evaporated to dryness under a nitrogen stream. The dried extracts of n-hexane (200 mg) and acetone (200 mg) were dissolved in 2 mL of chloroform. A total of 5 mg of different standards (cholesterol, campesterol, stigmasterol, βsitosterol, lupeol, betulin, and betullinic acid) (Sigma) was dissolved in 2 mL of chloroform.
Gas chromatography-flame ionization detection. Samples (2 μL) and standards (2 μL) containing cholesterol, campesterol, stigmasterol, β-sitosterol, lupeol, betulin, or betullinic acid were injected into the gas chromatography (GC) instrument. Acetate derivatization was per-formed by mixing ca. 1 mg of acetone extract with 2 mL of pyridine and 2 mL of an acetic acid anhydride. The mixture was incubated for 24 h in dark, evaporated into dryness under nitrogen, and then dissolved with 2 mL of ethyl acetate. The solution (2 μL) was then injected into the GC instrument. Gas chromatography-flame ionization detection (GC-FID) was performed with a capillary column 5% phenyl methyl siloxane (30.0 m × 320 µm × 0.25 µm) (Agilent Technologies 6890N) at a flow rate 40 mL/min (He) and FID 300°C. The column temperature was started at 220°C and then increased to 270°C at a rate of 10°C /min. Identification method. The EI-MS spectra of peaks 1-15 ( Figures 5 and 6) were compared with the EI-MS spectra of standards and EI-MS spectra from the Wiley database, the online databases of National Institute of Standard and Technology [14], the Spectral Database for Organic Compounds [15], MassBank [16], and published reports [17][18][19][20][21][22][23][24]. Peaks 1-15 were identified in accordance with the method of Commission Decision 2002/657/EC [25].

Results and Discussion
Microscopic identification and growth rate of CGS and F calluses. The CGS culture could be easily differentiated macroscopically from the F callus culture. The CGS culture showed a cohesive callus aggregate with diameters of 0.5-2 cm. By contrast, the normal callus culture was highly dispersed. Microscopic examination revealed that the formation of a thick cell wall limited the differentiation of the CGS culture. The thick cell walls of the CGC culture contained lignin as indicated by the development of a red color after the addition of phloroglucinol-HCl reagent. By contrast, F callus cultures lacked cell wall thickening and lignin ( Figure 2). The growth rate of CGS cultures was slower than that of the F callus culture (Figure 3).

Identification of sterol and triterpenes in CGS and F calluses.
Sterols and other triterpenes in the callus cultures were identified through TLC, GC-FID, and GC-MS analyses. The n-hexane and acetone extracts of CGS and F callus cultures were subjected to TLC analysis, visualized with anisaldehyde sulfuric acid spray reagent, and compared with standards. The results indicate that all n-hexane and acetone extracts contained sterol (β sitosterol) and triterpene (lupeol). However, betullinic acid was only detected in the acetone extract of callus culture (Figure 4). Further analysis was performed (A)  anisaldehyde sulfuric acid spray reagent, and compared with standards. The results indicate that contained sterol (βsitosterol) and triterpene (lupeol). However, betullinic acid was only detected in the acetone extract of the CGS callus culture (Figure 4). Further analysis was performed (B)

Mammosum Callus allus (A) and CGS Callus (B) After HCl ate of the F and CGS Callus Cultures
through GC-FID (Tables 1 and 2). The peaks of cholesterol, campesterol, stigmasterol, and β peaks at 31.678, 32.692, and 45.096 min were further identified through GCcycloartenol, and 24-methylene cycloartanol, respe tively. GC-FID analysis indicates that β the major compound in the n and CGS calluses, whereas βwere the major compounds in acetone extra and CGS calluses, respectively.   (Tables 1 and 2). The peaks of the cholesterol, campesterol, stigmasterol, and β-sitosterol peaks at 31.678, 32.692, and 45.096 min were further -MS as isofucosterol, methylene cycloartanol, respec-FID analysis indicates that β-sitosterol was the major compound in the n-hexane extract of the F -sitosterol and stigmasterol were the major compounds in acetone extracts of the F and CGS calluses, respectively.

Conclusion
The sterol profile of the CGS culture was more diverse than that of the normal callus culture. The production of several triterpenes, including betullinic acid, by the CGS culture but not by the normal callus culture could be attributed to differentiation by the CGS culture. This is the first report of betullinic acid from the in vitro callus culture of S. mammosum.