versión On-line ISSN 0717-9707
J. Chil. Chem. Soc. v.54 n.4 Concepción dic. 2009
J. Chil. Chem. Soc., 54, Nº 4 (2009), págs. 486-490.
CHEMOTAXONOMIC SIGNIFICANCE OF FLAVONOIDS IN THE SOLANUM NIGRUM COMPLEX
AYESHA MOHY-UD-DIN,*, ZAHEER-UD-DIN KHAN, MUSHTAQ AHMAD, MUHAMMAD AKRAM KASHMIRI, SAMMIA YASMIN, HINA MAZHAR
Centre for Plant Product Research, Department of Chemistry, GC University, Katchery Road, Lahore 54000, Pakistan. e-mail address: email@example.com
Five locally available plant taxa of Solanum nigrum Complex were investigated to resolve the International taxonomic controversy about these plants by analyzing their favonoid profles. Total favonoid contents in the taxa were determined by two complementary colorimetric methods, aluminum chloride method and 2,4-dinitrophenylhydrazine method. Thin layer chromatography was used to detect the glycosides of quercetin. Whereas GC-MS analysis of the hydrolyzed extracts showed the relative percentage of quercetin aglycon in the taxa under study. Statistical analyses of the results were employed for grouping of these taxa into various clusters. The signifcant distance found between S. americanum, S. chenopodioides, S. nigrum and S. villosum indicated them as distinct species. But S. retrofexum did not show such a marked difference and hence might be regarded as a variety or subspecies of S. nigrum.
Key words: Solanum nigrum Complex, Flavonoids, Chemotaxonomy.
Family Solanaceae comprises of about 84 genera and 3000 species1 and Solanum is one of its most important and largest genera. Solanum nigrum is the largest and most variable species of the genus Solanum and is now named as Solanum nigrum Complex because it is composed of a large number (about 30) of morphologically distinct taxa2. Plants are not only a source of food and fodder showing high nutritional value3 but are medicinally important as well4,5.
There have always been debates concerning the taxonomic complexity of species associated with the section Solanum6,7. The species related to S. nigrum have shown variations and similarities in various morphological aspects. This has obscured the morphological limits of the species and caused taxonomic confusion. For this reason, the species have been re-classifed many times but no satisfactory revision of the whole section has yet been devised. The boundaries between many of the species are still ill-defned, with many of the 'new' taxa proving to be no more than slight morphological variants of those already described. The situation has been further complicated by a number of authors, who have persistently treated different members of the section as belonging to one species, S. nigrum.
Three taxa belonging to S. nigrum Complex viz.: S. americanum Mill., S. nigrum L. and S. villosum Mill. had been reported in Pakistan by Schilling and Andersen2. S. chenopodioides Lam. and S. retrofexum Dunal are two other species that were found growing wild in and around the Botanical Garden, GC University, Lahore. On the basis of their morphological characters, classifcation of these taxa as being different varieties of S. nigrum or distinct species is controversial among the taxonomists. Morphological characters of these fve taxa are compared in Table 2. Each of these taxa has its own specifc medicinal and nutritional value8. Therefore, it is essential, for human health safety and quality control of the herbal medicine, to develop effcient methods for species identifcation/ delimitation. Their chemical analysis must be carried out to fnd out their relationship between them.
Flavonoids are widely occurring polyphenolic compounds and are extremely important because of their medicinal effects9. Flavonoids from different species of Solanum have been reported and reviewed10. Quercetin and its derivative glycosides make up most of the favonoid content in S. nigrum11. But the reports mostly did not take into account the morphological taxonomic complication associated with S. nigrum Complex and hence caused uncertainty. Flavonoids are useful secondary metabolites in assessing the relationship among closely related species or in studies of infraspecifc variation, and they are also occasionally useful in assessing phylogenetic relationships at higher levels12-14. The epicuticular components15, various terpenoids, sterols and favonoids have systematic signifcance and can be used for solving taxonomic problems16,17. GC-MS has been used in a number of occasions for the analysis of favonoids in plant. For example, in one study18, it was used to characterize the favonol aglycons in tomatoes (Solanum lycopersicum L.). In another study, the favonoid aglycons isolated from Propolis were identifed by GC-MS19. Also GC-MS was claimed to be useful in chemosystematics helping, for example, to characterize species on the basis of their cuticular wax15,20. We could not fnd any report on the analysis of favonoids of S. nigrum Complex by GC-MS and use of favonoids in its chemotaxonomy. So to search out the boundaries between these fve taxa using the chemotaxonomy to help resolve the International morphological taxonomic controversy on S. nigrum Complex we aimed to study their favonoid profles by comparing the total favonoid contents, quantitative comparison of quercetin aglycon by GC-MS along with qualitative study of their glycosides using TLC.
Plant samples of fve morphologically different plant taxa of Solanum nigrum Complex were grown under controlled conditions in Botanical Garden of GC University Lahore, Pakistan, each in specifed area and third accession of each was taken (approx. 1 Kg each) at fowering-seeding stage for chemotaxonomic investigation. Voucher specimens were authenticated and deposited in Dr. Sultan Ahmad Herbarium of GC University Lahore, Pakistan (Table 3).
Chemicals and Standards
Flavonoid standards Quercetin dihydrate and Naringin were purchased from MP. Biomedicals Inc. (Solon, Ohio). Methanol (HPLC grade), ethanol (100%), ethyl acetate, n-butanol, acetic acid, aluminum chloride (AlCl3), potassium acetate (CH3COOK), 2,4-dinitrophenylhydrazine (DNPH), potassium hydroxide (KOH), ammonia solution, hydrochloric acid (HCl) and sodium hydrogen carbonate (NaHCO3) were purchased from E-Merck (Darmstadt, Germany).
Sample preparation for Colorimetric analysis
Each of the plant samples was dewaxed by dipping in n-hexane for 45 sec21. Flavonoids were extracted for colorimetric analysis as described by Chang et al22. About 1 g (accurately weighed to 0.0001 g) each of dewaxed powdered plant sample was extracted with 25 mL of 95% ethanol under 200 rpm shaking for 24 hr. After fltration, the fltrate was adjusted to 25 mL with 80% ethanol and stored in an amber bottle.
(I)Aluminum Chloride Colorimetric Method: The aluminum chloride colorimetric method was modifed from the procedure reported by Woisky and Salatino23. Quercetin was used to make the calibration curve. Ten milligrams of quercetin was dissolved in 80% ethanol and then diluted to 25, 50 and 100 μg/mL. The diluted standard solutions (0.5 mL) were separately mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% AlCl3, 0.1 mL of 1M CH3COOK and 2.8 mL of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a Shimadzu UV-1700 Pharma Spec. spectrophotometer. The amount of 10% AlCl3 was substituted by the same amount of distilled water in a blank test. Then 0.5 mL of each ethanolic extracts with the AlCl3 solution for determination of favonoid content as described above.
(II) 2,4-Dinitrophenylhydrazine Colorimetric Method: The current method was modifed from the procedure described by Nagy and Grancai24. Naringin was used as the reference standard. Twenty milligrams of naringin was dissolved in methanol and then diluted to 500, 1000 and 2000 μg/mL. One milliliter of each of the diluted standard solutions was separately reacted with 2 mL of 1% 2,4-DNPH reagent and 2 mL of methanol at 50°C for 50 min. After cooling to room temperature, the reaction mixture was mixed with 5 mL of 1% KOH in 70% methanol and incubated at room temperature for 2 min. Then, 1 mL of the mixture was taken, mixed with 5 mL of methanol and centrifuged at 1,000 x g for 10 min to remove the precipitate. The supernatant was collected and adjusted to 25 mL. The absorbance of the supernatant was measured at 495 nm. The ethanolic extracts were similarly reacted with 2,4-DNPH for determination of favonoid content as described above.
Sample preparation for TLC and GC-MS analyses
30 g (10.0 g in a go) of dewaxed plant material was extracted in a Soxhlet apparatus with methanol (50 mL). At 1 hour intervals, aliquots were removed and checked for the presence of favonoids by TLC. After 5 hours, the extract showed absence of favonoids. The extraction procedure was executed in triplicate. Each extract was then fltered and the volume was completed to 100 mL with methanol. 10 mL from this extract was fltered through a small column of C18 silica (55-105 mm, 0.50 g) and the column eluted with 8 mL of methanol. The volume of the eluate was completed to 20 mL. More 5 mL of methanol was applied to the column and the eluate was checked for favonoids, which were absent. Total volume of each extract was made 25 mL.
TLC Procedure: Since quercetin, in its various glycosidic forms, has been the only favonoid detected in S. nigrum Complex, the preliminary examination of the above extract by TLC was carried out according to the solvent systems recommended for quercetin glycosides25. Normal phase TLC was performed on precoated (0.25 mm) silica gel 60 F254 sheets (Merck, Darmstadt, Germany). The plates were developed separately in 1: BAW (n-butanol-acetic acid-water, 4:1:5, top layer), 2: 15% Acetic acid (AcOH) in water. The chromatograms were observed in UV light (254 nm) before and after exposing to ammonia vapors. Experiment was repeated thrice and the results are given in Table 1.
Acid Hydrolysis: The standard procedure used for the hydrolysis of quercetin glycosides in Solanum lycopersicum L. has been described by Hertog et al26. HCl (1.2 M, 5 mL) was added in the methanolic extract (25 mL) of each sample and the mixture was stirred at 90oC under refux for 2 hours to obtain the aglycons by hydrolysis of favonoid glycosides. The extracts were cooled to room temperature and extracted with ethyl acetate (1:1, v/v). Fractionation with NaHCO3 was performed according to the method described by Sabatier et al27. The ethyl acetate extract was treated with 0.5 N NaHCO3 (1:1, v/v) three times to eliminate the free phenolic acids. The ethyl acetate extract was evaporated to dryness under a fow of nitrogen and favonols were re-dissolved in ethyl acetate. Each of the sample solution was fltered using 0.45µ polyamide flters (Sartorius, Germany) and was degassed by sonication for 3 min before injection. The fltrate (1 µL) was injected into GC-MS.
GC-MS analysis was carried out using the conditions modifed from the method of Tokusoglu et al18 used for the characterization of quercetin aglycon from hydrolyzed extract of Solanum lycopersicum L. GC-MS spectra were recorded on Shimadzu GCMS-QP2010A system in EI mode (70eV) equipped with a split/splitless injector (280oC), at a split ratio of 50/50 using DB-5MS column (30 m×0.25 mm i.d., flm thickness: 0.25 µm, J & W Scientifc, Fulsom, CA, USA). Helium was used as a carrier gas at the rate of 1mL/min. 1 µL of sample was injected keeping ion source temperature 200°C and interface temperature at 250°C. The column temperature was kept at 100°C for 1 min after injection and then increased at the rate of 10°C min-1 to 275oC which was held for 20 min. Standard stock solution 500 µg/mL of quercetin was prepared in methanol and the calibration curve was established using fve dilutions of the standard solution in the concentration range of 0.1-2.0 µg/mL. R2 value was 0.99.
Experiments were performed in triplicate and the results were expressed as ± Standard deviation (SD). Taxa under study were compared statistically on the basis of their favonoid profle by Minitab 13 Statistical Software using Euclidean distances for similarity determination and Multivariate cluster procedure.
RESULTS AND DISCUSSION
The diffculty of distinguishing genetically-controlled characteristics from phenotypic plasticity had long been known to impede species level taxonomy in Solanum9. The accessions of fve morphologically different plant taxa of S. nigrum Complex were taken at fowering-seeding stage for chemotaxonomic investigation. All the accessions were homogeneous and did not suffer from any pest or disease. Morphological comparison of the taxa is given in Table 1.
(I)AlCl3 Colorimetric method
Aluminium chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of favones and favonols. In addition it also forms acid labile complexes with the ortho-dihydroxyl groups in the A- or B-ring of favonoids28. Complexes were prepared and scanned at different wavelengths. In ring A, Ortho-dihydroxyl groups showed maximum absorbance at 415-440 nm, C-4 keto group at 385 nm, C-5 hydroxyl group and Ortho-dihydroxyl groups in B ring at 415 nm. In compromise, therefore, the wavelength 415nm was chosen for absorbance measurement. However absorbance of complexes formed by favanones such as naringin at this wavelength was too low to make the meaningful contribution to total absorbance. So these were estimated by 2,4-dinitrophenylhydrazine method. Quercetin is reported to be suitable for building the calibration curve22,23. Therefore standard Quercetin solutions ranging from 50 to 150 µg/ml concentrations were used to build up the calibration curve. The coeffcient of determination R2 was 0.953. Total amount of favones, favonols and isofavones was calculated and tabulated (Table 2).
(II)2,4-dinitrophenylhydrazine for determination of favanones
The principle of this method is that 2,4-dinitrophenylhydrazine reacts with ketones and aldehydes to form 2,4-dinitrophenylhydrazones. It was found that favones, favonols and isofavones with C2-C3 double bond could not react with 2,4-dinitrophenylhydrazine while the hydrazones of favanones showed maximum absorbance at 495 nm, and so this wavelength was selected for all measurements in the 2,4-dinitrophenylhydrazine reaction. Flavanone naringin, which was reported to show maximum absorbance at the above selected wavelength22, was used to make the calibration curve. R2 value was 0.96.
Total favonoid content
Total favonoid contents were represented as sum of two individual colorimetric methods. In fact, the total favonoid contents determined by HPLC can be greatly infuenced by the selected authentic standards and analytical conditions. Sometimes, limited by the availability of authentic standards, the identifcation of favonoid peaks in chromatograms may be incomplete. Therefore to stay away from preconception, we conducted the quantitative determination of favonoid contents in S. nigrum Complex by colorimetric analysis.
Results showed that favonoid contents of fve taxa as determined by aluminum chloride method, was much higher than those determined by 2,4-dinitrophenylhydrazine method (Table 2). The former ranged from 0.766 ± 0.012% to 1.616 ± 0.031% while the later ranged from 0.062 ± 0.005% to 0.5 ± 0.001%. As suggested by Chang et al22, the favones, favonols and isofavones formed complexes only with aluminum chloride, while favanones strongly reacted only with 2,4-dinitrophenylhydrazine. So the contents determined by the two methods were added up to evaluate the total content of favonoids. Results indicated that, among the fve taxa investigated, S. chenopodioides contained the lowest level of total favonoids (0.883 ± 0.020%), while S. nigrum showed highest level of total favonoids (2.116 ± 0.032%) (Table 2). Overall, there was great variation in total favonoid contents of the investigated taxa of S. nigrum Complex indicating that the quality of its medicinal use does require specifcation of the taxa taken.
TLC is a simple and reliable technique to compare the favonoid profles of different taxa and hence can be used to as an aid of their chemotaxonomy29. Various simple and complex glycosides of quercetin had been reported from S.nigrum Complex11,30. TLC of the extracts of the fve taxa under study displayed variability in their favonoid pattern (Table 3).
Two spots were identifed on the basis of their Rf values, color in UV light (254 nm) and UV light/ + ammonia25. Quercetin-3-glucoside (isoquercitrin) with Rf values 0.58 (BAW) and 0.37 (15% acetic acid) was detected in all the fve taxa. But quercetin-3-galactoside having Rf values 0.55 (BAW) and 0.35 (15% acetic acid) was detected only in S. nigrum and S. retrofexum. Color of both spots was yellowish brown in UV light (254 nm) but turned to bright yellow when examined in the same light after treating with vapors of ammonia. Since these glycosides had been previously identifed in different taxa of S. nigrum Complex11 and they are easy to detect by TLC, therefore, their presence in the samples can be indicated by comparing the observed properties with those reported in literature but other spots could not be characterized.
Characteristic occurrence of the favonoid glycosides helped comparing the taxa statistically. S. nigrum and S. retrofexum resembled much closely and were frst to segregate. This is because of presence of quercetin-3-galactoside in only these two taxa and much resemblance in their TLC pattern. However a spot (Rf: 0.36) was missing in S. retrofexum which can be attributed to difference at sub-species level. This cluster was distantly related to S. americanum which showed relatively less number of spots. But S. villosum and S. chenopodioides formed another cluster having almost same similarity index as of the previously discussed cluster (Fig. 1).
Quercetin in its different glycosidic forms has been the only favonol reported from S. nigrum Complex11. The presence of quercetin aglycon in the hydrolyzed extracts of the fve taxa under study was confrmed by GC-MS (Fig. 2).
Retention time of quercetin standard was 13.012 ± 0.001 min. It showed molecular ion peak of m/z (relative intensity in %) at 304 (22.8), base peak at 153 (100) with other characteristics peaks at 286 (4.0), 275 (27.6), 195 (2.8), 165 (12.4), 152 (24.4), 150 (21.2) and 123 (42.4). Quercetin in the samples was detected by spiking the samples with 0.5 µg/mL of standard quercetin solution and identifed on the basis of comparison with the retention time and mass fragmentation pattern of the standard, and of the mass spectrum with the data from NIST 147 Library linked to the mass detector. The extreme variation in its percentage composition in the extracts from the taxa grown, harvested and analyzed under similar conditions decides its part in their chemotaxonomy (Table 4). Comparing the area percentage, quercetin made up only 7.28% of the hydrolyzed favonoid extract of S. nigrum and this continued to increase irregularly while moving to S. retrofexum, S. villosum and S. chenopodioides. But S .americanum stand alone in the group with such a high percentage (92.92%). The calibration curve of standard was linear with R2 value of 0.99 in the concentration range of 0.1-2.0 µg/mL. Amount of quercetin varied from 3.06 ± 0.01 to 6.46 ± 0.01 mg/100g of plant (Table 4). Yang et al31 reported the quercetin content of S. nigrum and S. villosum. Our results were in agreement with this study for S. nigrum (3.7 mg/100g) but there was contradiction for S. villosum (18.1 mg/100g). However, no reports could be found for quantitative studies of quercetin on other three taxa under study.
Results were presented as mean ± SD (n=3). Determined by area normalization method. Calculated from the calibration curve.
The percentage variation of the favonoids observed in the fve taxa by colorimetric and GC-MS analyses were used to group them statistically. Here again S. nigrum and S. retrofexum formed a closely related cluster with a high similarity index showing much similarity in their favonoid profles. S. chenopodioides and S. villosum, although not so closely related to one another as compared to the previously discussed cluster, made another cluster. S. americanum aligned more distantly with above mentioned clusters. This is due to its very high quercetin concentration (shown by GC-MS analysis) and very low favanone percentage (in 2,4-dinitrophenylhydrazine method) as compared to other taxa (Fig. 3). This grouping pattern of these fve was similar to that obtained while comparing their epicuticular wax composition32. Burbank33 claimed to have derived S. retrofexum plant by hybridizing S. guineense and S. villosum that raised a controversy. But Heiser34 had proved that it was not the claimed hybrid derivative but a distinct species native to South Africa and Burbank may have inadvertently introduced it into his experimental garden and subsequently selected it as a new plant. Their many morphological differences particularly corolla color, stylar exsertion and color and shape of berries (Table 1) and our results contradict with Burbank claim.
With various biological activities, favonoids are the principal components in evaluating the quality as well as taxonomy of various taxa of S. nigrum Complex. The use of one colorimetric methods utilizing aluminum chloride reaction to determine favonoid contents was proved to be specifc only for favones and favonols, while another colorimetric method utilizing 2,4-dinitrophenylhydrazine reaction was specifc for favanones. Therefore, we used both analyses so that the sum of the results may better represent the real content of total favonoids. TLC was helpful in the preliminary examination of favonoid glycosides. GC-MS analysis enabled the quantitative comparison of quercetin aglycon. The results suggested that S. americanum, S. chenopodioides, S. nigrum and S. villosum had signifcant differences and might be treated as separate species and not the varieties/ subspecies of S. nigrum. In case of S. retrofexum, many similarities with S. nigrum were observed. So it could be regarded as the variety/ subspecies of S. nigrum. Some minor differences could be attributed to differentiation at variety/ subspecies level. Moreover S. retrofexum had many morphological variations as compared to S. villosum and showed no relationship with later in this study thereby rejecting the Burbank's claim to be its hybrid. Because of the taxonomic misunderstanding surrounding the component species and the tendency to refer to all members as 'S. nigrum', it is advisable that the information found in literature should be reinterpreted and any medicinal/commercial use of the taxa should be carried out in the light of above chemotaxonomic suggestion.
We acknowledge Mr. Zafar Siddiq Department of Botany, GC University Lahore, Pakistan, for providing plant materials. We are grateful to Dr. Mohsin Iqbal Department of Biotechnology, GC University Lahore, Pakistan, for his help in statistical analysis. This work was partly financed by Higher Education Commission Pakistan.
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(Received: April 14, 2009 - Accepted: August 24, 2009).