Abstract: Chemical constituents investigation of Gazania splendens Moore (Compositae family) let to isolate two flavonoids via luteolin and luteolin 7-O-glucoside, 11 n-alkanes (C14-C30), β-amyrin, cholesterol, β-sitosterol, stigmasterol and eight fatty acids. The structures of these compounds were established by Mass Spectrometry (MS), Gas-Liquid Chromatography (GLC) and spectroscopic techniques, including Ultra-violet (UV), Infra-Red (IR). The percentage of α-cellulose content in the leaves and flowers of the plant were 70.50 and 73.11%, respectively, hence the percentage in whole plant was 71.81%.
INTRODUCTION
Gazania splendens Moore belongs to the family Compositae which is one of the largest plant families, comprising 1,000 genera and 23,000 species. It is the source of relatively few products of economic and medical importance (Hutchinson, 1959). The most common species of genus Gazania are represented in Egypt by six species viz., Gazania hypridus, Gazania rigens R.Br., Gazania krebsiana Less, Gazania lichtenstenii Less, Gazania linearis Druce and Gazania splendens (Ahmed, 1981).
Uses in Traditional Medicine
To prevent miscarriage, relieve toothache, relieve earache, in cases of
stricture of the urethra (Ahmed, 1981 and Watt et al., 1962).
Previously Isolated Constituents
To the best of our knowledge there are no concrete studies on Gazania
splendens Moore, other than few studies which report the separation of dry
rubber, resin and some pigment compounds from flowers (Zechmeister et al.,
1943; Valadon et al., 1967; Bohlmann et al., 1979). In spite of
the phytochemical screening of Gazania species revealed the isolation
of xanthophylls, gazaniaxanthin, lutein, leafxanthophyll, γ-carotine, β-carotine,
lycopene, chrysanthemaxanthin, rubixanthin (Zechmeister et al., 1943;
Valadon et al., 1967), lupe-13-ene-3-acetate, lupe-13 ene-3-ol, lupeol,
lupe-20 (29) ene-3-acetate, two sesquiterpene lactones via gazaniolid, 8 α-isovaleryloxy-gazaniolid
(Bohlmann et al., 1979), alkaloids and flavonoids (Rizk et al.,
1986).
The aim of this research deals with the chemical study of the plant as regards their constituents particularly flavonoids, lipids, as well as study of α-cellulose content and proximate analysis.
MATERIALS AND METHODS
Plant Material
Fresh aerial parts of Gazania splendens MOORE were collected from
East and West Suez Canal region, Ismailia-Port Said road, Ismailia, Egypt in
April 2005 during the flowering stage. The identity was established by Prof.
Dr. Moustafa Zaghloul, Prof. of Floriculture and Medicinal plants, Department
of Horticulture, Faculty of Agriculture, Suez Canal University. A voucher specimen
(Number AMYM-1002) has been deposited in the Herbarium of Botany Department,
Faculty of Science, Suez Canal University, Ismailia, Egypt.
General Methods
Melting points were determined on Büchi 535 melting point apparatus.
IR spectra were recorded on 1430 Ratio Recording, Perkin-Elmer, in the Micro-analytical
unit, Faculty of Science, Suez Canal University (SCU). IR-data station Epson
FX-86e, in KBr disks. UV/Vis spectra were obtained on lambda 4B UV/Vis spectrophotometer
(Perkin-Elmer) in the region of 190-900 nm, in the Micro-analytical unit, SCU.
EIMS (ionization voltage 70 ev) was measured on GC-MSQP 1000EX Schimädzu,
in the Chemistry Dept., Cairo University (CU), Giza, Egypt. The fractions obtained
were subjected to gas-liquid chromatographic analysis (PYE UNICAM Series 304GC),
in Faculty of Agriculture, CU, using coiled glass column (2.8 m x 0.4 mm I.D.),
packed with Diatomite C (100-120 mesh) and coated with 1% OV-17, programmed
at 10°C/min from 70 to 270°C, then isothermally at 270°C for 25
min, injector temperature and FID detector at 300°C and the nitrogen carrier
gas at a flow rate of 30 mL min-1. GLC of the methyl esters of the
fatty acids was carried out by PYE UNICAM Series 304 Gas Chromatograph equipment
with FID and SGE injector split mode, using capillary column (25 m x 0.22 mm
I.D., 0.2 μm thickness) packed with vitreous silica coated with free fatty
acid phase, programmed at 12°C min-1 from 70 to 190°C, injector
temperature at 250°C, FID detector at 270°C and the flow rate of hydrogen
is 41.0 cm sec-1.
Extraction, Isolation and Characterization
The upper parts of the plant (leaves, flowers and stems) were air dried
and ground altogether as a fine powder. The phytochemical screening and the
proximate analysis included ash and moisture contents were performed in accordance
with AOAC (1990) and Balbaa, (1976).
Acid-insoluble Lignin
The acid-insoluble Lignin of leaves and flowers were determined using Tappi
Standard Methods (1954).
Determination of Cellulose Content
Hollocellulose and α-cellulose contents were calculated according to
Wise et al., (1946) and Tappi Standard Methods (1954).
Lipids
Air dried and powder aerial parts (2 kg) of the plant were extracted with
petroleum ether (40-60°C) to yield 60 g of a dark green oily residue (lipid
fraction). The marc was macerated with ethanol (80%) at room temperature till
exhaustion. The resulting alcoholic extract was concentrated in vacuo to obtain
a crude residue (950 g). About 35 g of the obtained lipid fraction were purified
by treatment with fuller’s earth. The purified residue treated in accordance
with (Berry, 2004) to effort 4.5 g of acetone insoluble fraction and 18 g of
acetone soluble fraction as oily material. Ten grams of the acetone soluble
fraction were saponified to yield 4.4 g of yellowish brown, semi-solid residue
of unsaponifiable matter and 3.8 g of semi-solid residue of fatty acids. Three
grams of the unsaponifiable matter were subjected on a silica gel column chromatography
(80x2 cm) with petroleum ether and increasing the polarity with benzene.
Flavonoids
The flavonoids were isolated by treating the alcoholic extract of the plant
with Hyflo super cel, then evaporated till dryness. The obtained homogeneous
powder was packed into a glass column (diameter 5x60 cm) eluted with petroleum
ether (40-60°C), followed by extraction with chloroform (CHCl3)
then ethyl acetate, to effort 7.5 and 11.5 g, respectively. Paper Chromatography
(PC) and Preparative Paper Chromatography (PPC) were carried out in accordance
with (Jung et al., 2004). Detection was carried out using UV-light at
366 nm before and after exposure to ammonia vapor and spraying with Naturstoff
reagent-A (Neu, 1956). Fractionation of the flavonoids was affected by column
chromatography. Five grams of the ethyl acetate extract were applied on a glass
column packed with silica gel G. (60 cm x 3 cm I.D.) in CHCl3. Elution
was carried out with CHCl3, followed by CHCl3/ MeOH with
an increasing amounts of the later till 80%. Moreover, application of sephadex
LH-20 column chromatography was applied for the separation and purification
of the flavonoids, elution was affected with 90% MeOH. The purity of the isolated
components G-I and G-II was checked by two-dimensional TLC on a cellulose plate.
UV- absorption spectra of the isolated flavonoidal components were measured
in MeOH as well as in MeOH after the addition of shift reagents (Mabry et
al., 1970).
Acid Hydrolysis of the Flavonoidal Compound (G-II)
Acid hydrolysis of Compound (G-II) (25 mg) was carried out in accordance
with Brown and Rice-Evans, (1998).
Compound (G-I). (55 mg), m.p. 327-329°C, Rf; [0.54, BAW (4:1:5)]. IR bands (KBr) vmax: 3417, 1657, 1616, 1579, 1515, 1397, 1313, 1266, 1190, 1165 and 1120 cm-1. UVλmax nm (MeOH): 245sh, 255, 268.7, 292sh, 350.7. EIMS m/z (rel. Int.): 286 (100), 258 (14.6), 153 (30.5), 152 (7.1), 137 (2.8), 134 (12.9), 109 (1.0).
Compound (G-II). (70 mg), Rf; [0.34, BAW (4:1:5)]. IR bands (KBr) vmax: 3303-3362, 1663, 1618, 1571, 1520, 1515, 1388, 1260 and 1181 cm-1. UVλmax nm (MeOH): 255.6, 268.7sh, 349.7, 400sh. EIMS m/z (rel. Int.) (after hydrolysis): 286 (100), 258, 153, 152, 137, 134, 109.
RESULTS AND DISCUSSION
The results obtained from the preliminary phytochemical screening of Gazania splendens Moore revealed the presence of flavonoids coumarins, carbohydrates and/or glycosides, sterols and/or triterpenes and tannins. The average percentages of the constants of the plant were calculated as shown in Table 1.
The percentage of α-cellulose content in the leaves and flowers of the plant were 70.5 and 73.11% respectively, as shown in Table 2. The percentage in whole plant was 71.81%. This percentage represented approximately twice the percentages present in each of Bagasse (40%) and Rice straw (38%) (Anonymous, 2000; Chan, 1999), The amount of dried leaves of the plant from feddan, in one season was 15.864 ton. While the amount of dried flowers of the plant from feddan, in one season was 3.890 ton, this shows the economic importance of the Gazania splendens plant as a source for paper manufacturing industries.
| Table 1: | Constants of Gazania splendens Moore |
| iiCalculated on the air-dried plant material, iiiAverage Percentages (means of duplicate analysis), ivCalculated on the oven-dried material | |
| Table 2: | α-cellulose content of Gazania splendens Moore |
| Table 3: | Gas liquid chromatographic analysis of the hydrocarbon fraction |
| in: normal | |
The results obtained from the GLC chromatogram of hydrocarbon fraction, eluted with petroleum ether-benzene (90:10), revealed the presence of a series of 11 n-alkanes (n-C14 to n-C30) with C22H46 and C30H62 as the major constituents of the mixture 21.04 and 19.57 % respectively and the percent of the other constituents ranging from 3.53-12.69% as shown in Table 3 and Fig. 1.
The residue obtained from fractions eluted with petroleum ether-benzene (75:25) white crystalline needles in MeOH. It melted at 197-199°C both alone and upon admixture with authentic β-amyrin. It gives positive Liebermann-Burchardt reaction, indicating that it is a triterpenoid in nature. TLC on Silica gel using different solvents showed to be a single spot possessing the same retention time as authentic β-amyrin. The IR-spectrum of the isolated substance showed the same absorption bands characteristic for authentic β-amyrin.
The combined fractions eluted with petroleum ether-benzene (25:75) gave white crystalline needles in CHCl3/ MeOH, m.p. 133-135°C. The substance showed a single spot on TLC, Silica gel G, using toluene- acetone system (90:10), possessing Rf; 0.47. It was identical with authentic β-sitosterol. However, several authors (Bennet and Heftman 1966; Marsill and Moreill 1968; Hornero-Mendez and Minguez-Mosquera, 2000; Singh et al., 1970) reported that the sterols isolated from plants, showed single substances on TLC and their physical properties confirmed this. Yet on subjecting to GLC they were proved to be mixtures. So the sterol fraction was subjected to GLC. The obtained chromatogram revealed that, the isolated sterol fraction is a mixture of three compounds possessing the same retention times (18.69, 19.85 and 20.04%, respectively) as authentic Cholesterol, β-sitosterol and Stigmasterol. The percentages of them were 17.3, 39.2 and 43.5% respectively from the total sterol fraction. GLC chromatogram of the methyl ester of fatty acids revealed the presence of myristic, pentadecanoic, palmitic, heptadecanoic, stearic, oleic, linoleic and linolenic acids according to their retention times in comparison with authentic (3.06, 0.38, 67.89, 0.37, 1.52, 2.87, 19.61 and 4.30%, respectively).
| Fig. 1: | GLC chromatogram of hydrocarbon fraction |
The saturated fatty acids represent 73.22% while the unsaturated fatty acids represented by 26.78% of the total fatty acids. The major fatty acids were palmitic acid 67.89% and linoleic acid 19.61% as shown in Table 4 and Fig. 2.
TLC and PC of the ethyl acetate extract were performed better separation and revealed the presence of four flavonoid constituents, two major flavonoid Rf; (0.54, 0.34; BAW 4:1:5) and two minor flavonoid constituents Rf; (0.86, 0.77; BAW 4:1:5) beside fluorescent constituent Rf; (0.61; BAW 4:1:5). On the other hand, the chloroform extract was found to contain two flavonoidal spots Rf (0.54, 0.34, BAW 4:1:5) as minor constituents. Compound (G-I). UV-spectrum of (G-I) in MeOH (Table 5 and Fig. 3), exhibited λmax at 350.7 nm (band-I). This indicates that it is a flavone type. The NaOMe spectrum showed a bathochromic shift of 52.5 nm in band-I, which is indicated to the presence of 4`-OH group.
| Table 4: | Gas liquid chromatographic analysis of the methyl esters of fatty acids fraction |
| Fig. 2: | GLC chromatogram of saponifiable fraction |
Also, showed a shoulder peak at 328 nm which is an indicative to the presence of 7-hydroxy group. AlCl3 spectrum showed peak > 415 nm (426.2 nm) and AlCl3/HCl spectrum showed peak I<395 nm (386.8 nm). This indicates that the substance G-I belongs to the phloroglucinol type A ring (i.e., 5, 7-dihydroxy) (Voirin, 1983).
| Fig. 3: | UV/Vis absorption spectra of compound G-I in (a) MeOH and Me OH/NaOMe, (b) MeOH, MeOH/AlCl3 and MeOH/AlCl3/HCl and (c) MeOH, MeOH/NaOAc and MeOH/NaOAc/H3BO3 |
Also, AlCl3 spectrum showed a relatively large bathochromic shift (75.5 nm) indicating the existence of ortho-dihydroxy system and/or 3, 5-di-hydroxy groups. However, AlCl3/HCl spectrum exhibited hypsochromic shift (39.4 nm) in band-I relative to AlCl3 spectrum. This performed the presence of 3', 4'- ortho-dihydroxy groups. The presence of only a free 5-hydroxyl group is mainly due to the persistence of a bathochromic shift (36.1 nm) in band-I of AlCl3/HCl spectrum (relative to MeOH spectrum) and not due to 3-hydroxyl group. NaOAc spectrum showed a bathochromic shift (18 nm) in band-II, indicates the presence of free 7-OH group. An ortho-dihydroxy system is further proved to exist in ring-B by the bathochromic shift (44.3 nm) in band-I of the NaOAc/H3BO3 spectrum. IR-spectrum of G-I showed a strong band at 3417 cm-1 corresponding to -OH group, a strong band at 1657 cm-1 corresponding to an α-β-unsaturated ketone (C = O group of (γ-pyrone), absorption bands at 1616, 1579, 1515 cm-1 (an aromatic system); in addition to the absorption bands at 1397, 1313, 1266, 1190, 1165 and 1120 cm-1. MS of G-I showed a molecular ion (M+) at m/z 286 (100) which corresponds to the molecular formula C15H10O6 of four hydroxy substitution pattern. Hence, the fragmentation pathway undergoes the Retero-Diels Alder reaction-giving rise to ring-A fragment at m/z 153 (30.5) and m/z 152 (7.1). However, the hydrogen transfer ion at m/z 153 is much intense than that of the normal fragment ion at m/z 152, indicating that it has 5,7-di-hydroxy grouping. Moreover, the ring-B fragment at m/z 134 (12.9) indicates that the other two-hydroxyl groups must be attached to ring-B. Furthermore, loss of CO directly from the molecular ion (M+-CO) was also shown, leading to the phenylbenzofuran fragment ion at m/z 258 (14.6) which further fragments giving rise to the benzoyl ion at m/z 137 (2.8). This loses CO directly giving m/z 109 (1.0). The previous data are in agreement with those reported for luteolin (Johnson et al., 2002; Ðordević et al., 2000; Braune et al., 2001; Wouters Rosario-Chirinos, 1992; Geissman, 1955; Mabry et al., 1970; Macek, 1972), (Fig. 4).
Compound (G-II). UV-spectrum in MeOH, exhibited λmax at 349.7 nm (band-I), which indicates that it is a flavone type. The NaOMe spectrum showed a bathochromic shift of 53.5 nm in band-I with slow degeneration indicated to the presence of 4`-OH group. AlCl3 spectrum showed peak > 415 nm (427.8 nm) and AlCl3/HCl spectrum showed also peak I < 395 nm (388.4 nm) (Fig. 5). This indicates that the substance G-II belongs to the phloroglucinol type A ring (i.e., 5, 7-dihydroxy) (Voirin, 1983).
| Table 5: | UV/Vis absorption maxima of the isolated flavonoidal constituents |
| iish: shoulder | |
| Fig. 4: | Luteolin; R = OH, Luteolin 7-O-glucoside; R = O-glu |
| Fig. 5: | UV/Vis absorption spectra of compound G-II in (a) MeOH and MeOH/NaOMe, (b) MeOH, MeOH/AlCl3 and MeOH/AlCl3/HCl and (c) MeOH, MeOH/NaOAc and MeOH/NaOAc/H3BO3 |
Also, AlCl3 spectrum showed a relatively large bathochromic shift (78.1 nm) indicating the presence of ortho-dihydroxy system and/or 3 and/or 5-hydroxy groups at ring B. AlCl3/HCl spectrum exhibited hypsochromic shift (39.4 nm) in band-I relative to AlCl3 spectrum, which performed the presence of 3', 4'- ortho-dihydroxy groups. The presence of only a free 5-hydroxyl group is mainly deduced from the persistence of a bathochromic shift (38.7 nm) in band-I of AlCl3/HCl spectrum (relative to MeOH spectrum) and not due to 3-hydroxyl group. No bathochromic shift was observed in band-II in the NaOAc spectrum, indicates the absence of free 7-OH group and it may be occupied by the sugar moiety. Furthermore, the presence of free 7-hydroxyl group in its aglycone after acid hydrolysis was confirmed by the bathochromic shift (13.1 nm) in band-II of NaOAc spectrum. IR-spectrum of G-II showed a strong band at 3303-3362 cm-1 corresponding to -OH group, a strong band at 1663 cm-1 corresponding to a α, β-unsaturated ketone (C = O group of (γ-pyrone), absorption bands at 1618, 1571, 1520 and 1515 cm-1 (an aromatic system); in addition to the absorption bands at 1388, 1260 and 1181 cm-1. MS of G-II after hydrolysis, showed a molecular ion (M+) at m/z 286 (100), which corresponds to the molecular formula C15H10O6 of parent flavone 222 plus four hydroxy substitution patterns 64. The mass spectral data are in agreement with those reported for luteolin. The previous data are in agreement with those reported for luteolin 7-O-glucoside as shown in Fig. 4.
The Rf-values as well as the color under UV-light showed that, it is probably a flavonoidal glycoside in nature. PC analysis of G-II residue after hydrolysis revealed the presence of glucose as a sugar moiety while the aglycone was identified as luteolin by comparison with authentic reference. The previous data are in agreement with those reported for luteolin 7-O-glucoside (Godjevac et al., 2004; Nissler et al., 2004; Kader, 2006; Geissman, 1955; Mabry et al., 1970; Macek, 1972).
CONCLUSION
The high α-cellulose content approximately twice that of Bagasse (40%) and Rice straw (38%), shows the economic importance of the Gazania splendens plant as a source for paper manufacturing industries. Investigation of Gazania splendens resulted in the isolation and identification of flavonoids and lipid constituents with percentages 2.4 and 3.0% relative to the total powder plant. The flavonoid constituents include luteolin and luteolin 7-O-glucoside as the major constituents. The isolation and identification of lipid fraction resulted in 11 n-alkanes (C14-C30), β-amyrin, cholesterol, β-sitosterol, stigmasterol and eight fatty acids. The saturated fatty acids represent 73.22 % while the unsaturated fatty acids represented by 26.78% of the total fatty acids. The major fatty acids were palmitic acid 67.89% and linoleic acid 19.61%.
ACKNOWLEDGMENT
We thank Prof. Dr. Moustafa Zaghloul, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt for his kind help in identification of the plant.