Abstract: Four flavonoidal compounds identified as quercetin, isorhamnetin-α-3-O rhamnoside, quercetin 3-O-β-D-glucopyranosyl -(1''-6''')-β-D-glucopyranoside and quercetin 3-O-β-D-galactopyranosyl -(6''-1''')- α-L-2''' acetyl rhamnose-(3'''-1'''') β-D-glucopyranoside were for the first time isolated from the ethyl acetate and n-butanol soluble fractions of the alcoholic extract of Fagonia indica Burm F. in addition to oleanolic acid, β-sitosterol-3-O-β-D-glucoside and stigmasterol 3-O-β-D-glucoside. Flavonoids amounted to 3% as estimated colorimetrically by using aluminum chloride. The analgesic activity of the alcoholic extract of Fagonia indica Burm F. was tested by the writhing and the hot-plate tests using acetyl salicylic acid (200 mg kg-1, i.p.,) and morphine (10 mg kg-1, i.p.,) as reference drugs. The cytotoxic and the antimicrobial activities of the alcoholic extract as well as its fractions were also determined. The alcoholic extract elicited activity on both acetic acid-induced writhing response as well as hot plate test in mice showing its central and peripheral mediated antinociceptive action. The alcoholic extract as well as its fractions exhibited marked antitumor activity against the two cell lines tested with the ethyl acetate fraction showing the least IC50 against both carcinoma cells MCF7 and HCT cells while the chloroform fraction showed highest antimicrobial activity against Pseudomonas aeruginosa.
INTRODUCTION
Genus Fagonia includes about 35 species that are distributed in the deserts and dry areas in India, tropical Africa, Chile and South West USA. Many of the species are spiny herbs or sub-shrubs (Chopra et al., 1982). Fagonia species are reported to be medicinal in the scientific literature as well a s in folk medicine. It was reported that some species have cytotoxic (Ibrahim et al., 2007) anti-microbial (Gehlot and Bohra, 2000) and antihypertensive (Gibbona and Oriowo, 2001) activities. These activities were attributed to the presence of a variety of active ingredients including triterpenoidal saponins (Abdel Khalik et al., 2000), flavonol glycosides (Lamyaa et al., 2008) as well as acids like ursolic and oleanolic acids either alone or with their derivatives (El-Wakil, 2007).
Fagonia indica Burm F. (Mushikka or white spine) is a plant distributed in the deserts of Asia and Africa (Beier et al., 2004). It is used in folk medicine for cancer as well as most of the disorders considered to be due to poisons. Survey of literature revealed few reports indicating the presence of saponins and oleanolic acid in Fagonia indica Burm F. (Shaker et al., 1999; Yeung and Che, 2009) and almost no reports about isolation of the flavonoidal constituents. Moreover, only few studies investigated the pharmacological effects of the plant extract (Soomro and Jafarey, 2003; Mandeel and Taha, 2005) with no reports about its possible analgesic effect or antitumor activity using two cell lines (breast and colon).
The aim of the present study therefore is to determine the constituents of the alcoholic extract of Fagonia indica Burm F. to evaluate the acute toxicity of the plant extract as a measure of its safety and to investigate the possible antinociceptive as well as the anti-tumor activities of the total alcohol and the fractions prepared. This is considered as a new challenge since no published data explored this area of research.
MATERIALS AND METHODS
Plant material: The whole plants of Fagonia indica Burm F. were collected during July till August (2008) from desert plants growing in Dubai, United Arab Emirates, while the whole research study has been performed during the period from July to the end of November (2008). Identification was kindly verified by Dr. Hassnaa Ahmed Hosny, Professor of Plant Taxonomy, Department of Botany, Faculty of Science, Cairo University. Vouchers specimens were kept at the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Egypt.
Equipments for phytochemical investigation: TLC was carried out on pre-coated silica gel 60 F254 plates (E.Merck). Stationary phases used for column chromatographic fractionation viz., Silica gel G 60, RP-18 (E. Merck) and Sephadex LH-20 (Sigma- Aldrich). M.P. was measured on a Digital melting point apparatus (Electrothermal IA 9000 series) and was uncorrected. UV absorption spectra were run on a Shimadzu 1700 spectrophotometer. 1H-NMR spectra were obtained at 500 MHz and 13 C -NMR spectra at 125 MHz on JEOL GX-500 spectrometer with the chemical shifts (δ ppm) expressed relative to TMS as internal standard.
Animals: Male albino mice, weighing 20-25 g were provided by the Pharmacology Department, Dubai Pharmacy College. They were located in a special well-equipped atmosphere and had free access to food and water. They are left for a period of one week for accommodation before performing the experiments.
Drugs, chemicals and solvents: Acetyl salicylic acid and naloxone hydrochloride were purchased from Sigma, USA; morphine hydrochloride from E. Merck, glacial acetic acid from Fisher Scientific, USA; sodium bicarbonate and all organic solvents used were obtained from El-Nasr Chem. Co., Cairo, Egypt.
Microorganisms:
| • | Gram positive bacteria: | Staphylococcus aureus and streptococcus beta hemolytica |
| • | Gram negative bacteria: | Escherichia coli, Pseudomonas aeruginosa and proteus |
| • | Fungi; Candida albicans: | All were obtained from Microbiology Department, Dubai Medical College |
Anti-tumor cell lines: Mammary carcinoma F7cells (MCF7) and Human colon tumor cells (HCT) were purchased from National Cancer Institute, Cairo University, Egypt.
Preparation of the different plant extracts: Alcoholic extract of the plant under investigation was obtained by exhaustive cold maceration of air-dried powdered plant (1.370 kg) in ethanol (70%). The alcoholic extract was then evaporated to dryness, under vacuum, at a temperature not exceeding 50°C. The solvent-free residue (40.0 g) was suspended in water and fractionated by successive extraction with n-hexane, chloroform, ethyl acetate and n-butanol saturated with water. The extracting solvent, in each case, was removed by vacuum distillation. For biological study, the different extracts were reconstituted with distilled water or with tween 80.
Phytochemical study
Colorimetric estimation of the flavonoid content: One gram of air-dried
defatted powdered plant was subjected to quantization of their flavonoid content.
The method adopted is based on measuring the intensity of the yellow color developed
when flavonoids are mixed with aluminum chloride (Mabry et
al., 1970). The percentage of flavonoids was calculated as quercetin
with reference to a pre-established standard calibration curve using different
concentrations (ranging from 8-200 μg 5 mL-1) of 0.04% w/v ethanolic
solution of quercetin. Results obtained were the average of triplicate experiments.
Investigation of the chloroform extract: A portion (7.1 g) of the chloroform fraction was applied on VLC column (25x5 cm). Gradient elution was performed with hexane-chloroform, chloroform-ethyl acetate mixtures. Fractions, 100 mL each, were collected and monitored by TLC (solvent systems, chloroform methanol 9.7:0.3 and 9:1, spots visualization, by examination under UV before and after exposure to NH3 vapor and by spraying with P-anisaldehyde). Those shows the same chromatographic patterns were pooled yielding five collective fractions (Fc1-Fc5).
Fc4 (Column system, chloroform-ethyl acetate, 85:15; 0.5 g and showed 5 spots with RF values 0.77, 0.70, 0.64, 0.60 and 0.50) and Fc5 (Column system, chloroform-ethyl acetate, 70:30; 0.61 g and showed 6 spots with RF values 0.60, 0.41, 0.40, 0.23, 0.15 and 0.09) were subjected to refractionation on Reversed Phase Silica (RP-18) columns by using methanol-water mixtures, afforded 3 compounds S1-S3, respectively.
Investigation of the ethyl acetate and n-butanol extracts: Portions (3.99 and 6.3 g) of the ethyl acetate and butanol extracts respectively were fractionated on Sephadex LH-20 column (52.5x2.5 cm). Gradient elution was performed with water-methanol mixtures followed by methanol. Fractions (50 mL each) were collected and monitored by TLC using solvent system chloroform-methanol 8.5:1.5 and 8:2; spots were visualized by examination under UV before and after exposure to NH3 vapor and by spraying with p-anisaldehyde. Similar fractions were pooled to yield collective fractions (Fe1-Fe4 from ethyl acetate and Fb1-Fb4 from butanol extracts). Fe2 (coming f rom Sephadex column system, methanol-water 50:50; 0.56 g, showed 2 spots with RF: 0.37 and 0.11) and Fe3 (coming from Sephadex column system, methanol- water 60:40; 0.27 g, showed 3 spots with RF: 0.60, 0.50 and 0.37) and Fb2 (coming from sephadex column system, methanol-water 8:2; 0.6 g; and showed 3 spots with RF : 0.83, 0.45 and 0.18) were subjected to refractionation, on Sephadex LH- 20 columns by isocratic elution using methanol -water 8:2, afforded 4 compounds S4-S7, respectively which gave positive tests for flavonoids.
The identification of all isolated compounds was based on physicochemical and spectral analysis as well as on comparison with reference samples and published data.
Biological study
Acute toxicity testing of the total plant alcoholic extract: The
acute toxicity of the extract was tested in mice by both oral and intraperitoneal
routes. Several doses of the extract were chosen in a manner allowing for equal
logarithmic intervals between the different doses. Accordingly, the chosen doses
were 32, 64, 128, 256, 512, 1024, 2048 and 4096 mg kg-1 body weight
to different groups of mice, each consisting of 10 animals. For oral toxicity
testing, food was withheld from the animals overnight before administration
of the extract. The extract was freshly prepared by reconstitution in distilled
water. Mortality was assessed over two consecutive days and animals were observed
for any illness or abnormal behavior.
Antinociceptive activity of the total plant alcoholic extract
Writhing test: Thirty five mice have been selected and divided into
5 groups consisting of 7 animals each. Three groups were treated with the alcoholic
extract of Fagonia indica Burm F. at doses (2, 5 and 10 mg kg-1).
Aspirin at a dose of 200 mg kg-1 (Sarra et
al., 2005) or normal saline for control were injected i.p. to the rest
2 groups of mice. Injection was carried out 30 min before induction of writhing.
Trial of higher doses of the extract (25, 50 and 100 mg kg-1) has
been also carried out (data not shown). The test was conducted according to
that described by Koster et al. (1959). The writhing
response was elicited by an i.p. injection of 0.1 mL 0.6% acetic acid. Animals
were then placed individually into glass beakers and 5 min were allowed to elapse
before they were observed for 10 min. The number of writhes during 10 min was
recorded for each animal. Significant reduction in the number of writhes compared
to the control was considered analgesic response.
Hot-plate test: Two sets of experiments have been carried out. Each set consisted of five groups of mice of 8 animals each. In the first set, the first group was given normal saline and served as control group w hile the other four groups were i.p., injected with either morphine at a dose of 10 mg kg-1 (Natorska and Plytycz, 2005) or increasing doses of the plant alcoholic extract (2, 5 and 10 mg kg-1). In the second set, animals were subjected to the same treatment regimens as in the first one except that the mice in groups 2-5 were injected with naloxone at a dose of 2 mg kg-1, i.p., (Wibool et al., 2008) 15 min before administration of the stated agent. All mice were subjected to the hot-plate test before any treatment, then, the stated agents were given in a total volume of 0.2 mL. The hot-plate test was performed at 30 and 60 min thereafter. The test was conducted according to that described by Eddy and Leimbach (1953). Mice are brought to the testing room and allowed to acclimatize for 10 min before the test begins. Pain reflexes in response to the thermal stimulus are measured using a Hot-Plate Analgesia Meter (Ugo Basile, Varese, Italy). The surface of the hot plate is heated to a constant temperature of 55±0.5°C, as measured by a built-in digital thermometer. Mice are placed on the hot plate which is surrounded by a clear acrylic cage and the latency to respond with either a hindpaw lick, or jump is measured to the nearest 0.1 second. The mouse is then immediately removed from the hot plate. A maximum cut-off period of 30 seconds was applied to avoid nerve damage. Animals are tested once at a time and are not habituated to the apparatus prior to testing.
Statistical analysis: For results of the writhing experiment, data were analyzed by Kruskal-Wallis non parametric one-way ANOVA followed by Dunn's multiple comparison test. For results of the hot-plate experiment, data were analyzed by one-way ANOVA followed by Tukey-Kramer multiple comparison test (Armitage and Berry, 1987). For both statistical procedures, differences at p<0.05 were considered significant.
Screening for anti-microbial activity towards selected microorganisms: The alcohol and the extracts prepared from it were subjected to in-vitro screening for antimicrobial activity. The samples were tested at dose of 200 mg mL-1 using Tween 80 to assist solubility. Selected strains of bacteria and fungi were used adopting the agar diffusion technique cup method for bacteria and fungi (Lorian, 1991). The effect of the different samples were compared with broad spectrum antibiotics; Penicillin 10 mg and Doxycycline HCl 30 mg. Evaluation of the antimicrobial activity was based on measuring the diameters of the observed zones of the inhibition in mm.
Anti-tumor activities of the total plant alcoholic extract, the ethyl acetate and the butanol fractions: Potential cytotoxicity of the total alcoholic extract and the ethyl acetate and butanol fractions prepared from the parent alcoholic extract, were tested using the method of Skehan et al. (1990). Intensity of the color was measured at λ570 nm in an ELISA reader. The percentage of surviving fraction of each tumor cell line was recorded for each concentration of the different extracts tested.
RESULTS AND DISCUSSION
Phytochemical study
Colorimetric estimation of the flavonoid content: The percentage
of flavonoids as determined by AlCl3 method for the whole plant was
found to be 3.5%.
Constituents of the chloroform extract
Compound S1: white needles crystals, 20 mg, RF
value 0.5, m.p. 310-312°C (methanol) was soluble in chloroform-methanol
mixture. It exhibited the following spectra data: 1H-NMR, δ(500
MHz, DMSO): 0.77, 0.79, 0.81 0.93, 0.94, 1.0, 1.20, (7x CH3) 3.94
(1H, m, H-3), 5.3 (1H, t, olefinic H-12).
Compound S2: white needle crystals, 50 mg, RF value 0.41; m.p. 140-143 °C (methanol) was soluble in chloroform-methanol mixture. It exhibited the following spectra data: 1H-NMR, δ(500 MHz, DMSO): 0.75 (3H, s, Me-18), 0.78 (3H, d, J = 7.2 Hz, H-26), 0.80, (3H, d, J = 7.2 Hz, H-27), 0 .87 (3H, t, J = 8.4 Hz, H-29), 0.94 (3H, d, J = 6 Hz, H-21), 1.20 (3H, s, H-19), 3.94 (1H, m, H-3), 4.66 (1H, d, J = 8Hz, H-1π, Glc.), 5.36 (1H, s, H-6).
Compound S3: white needles crystals, 30 mg, RF value 0.40, m.p. 170°C (methanol) was soluble in chloroform-methanol mixture. It exhibited the following spectra data: 1H-NMR, δ(500 MHz, DMSO): 0.60 (3H, s, Me-18), 0.81 (3H, d, J = 7.2 Hz, H-26), 0.88, (3H, d, J = 7.2 Hz, H-27), 0.92 (3H, t, J = 8.4 Hz, H-29), 1.2 (3H, d, J = 6 Hz, H-21), 1.50 (3H, s, H-19), 3.94 (1H, m, H-3), 4.40 (1H, d, J = 8Hz, H-1π, Glc.), 5.21 (2H, m, H-23), 5.40 (1H, s, H-6).
13C-NMR data for compounds S1-S3 was present in Table 1.
The 1H-NMR spectrum of compound S1 showed 7 methyl groups at δH 0.77 0.79, 0.81, 0.93, 0.94, 1.0 and 1.2. The signal at δH 5.3 was due to the olefinic proton at C-12 while the proton germinal to the hydroxyl group was observed at δH 3.94. From the 1H-NMR and 13C-NMR and in accordance with the published data (Adesina and Reisch, 1985), Compound S1 is identified as oleanolic acid.
| Table 1: | 13C-NMR data (125 MHz, δ ppm) of compounds S1-S3 isolated from chloroform extract of Fagonia indica Burm F. |
The 1H-NMR spectra of compounds S2 and S3 demonstrated the characteristic features of a steroidal nucleus, this is in accordance with the previous findings of Goad and Toshihiro, (1997). Both compounds showed presence of 6 methyl groups. The two methyl singlet at δH 0.6-0.75 and 1.20 were assigned for H-18 and H-19 respectively. The three doublets at δH 0.78-0.81, 0.80 -0.88 and 0.94-1.20 were attributed to H-26, H-27 and H-21, respectively, while the triplet at δH 0.87-0.92 assigned to H-29, which is characteristic for C-24 ethyl sterols. In addition, compound S3 showed multiplet at δH 5.21 assigned to H-23. The anomeric proton that appeared at δH 4.40-4.66 with J = 8 Hz indicated a β-linkage. The 13C-NMR revealed the presence of 35 signals, 29 of which were assignable for the C-24 ethyl sterols nucleus (Table 1) and the other 6 signals corresponded to the sugar moiety. The olefinic carbons at C-5 and C-6 were verified by the presence of signals at δc 140.1 and 121.8 in both compounds S2 and S3. Addition olefinic carbons in compound S3 at C-22 and C-23 were verified by the presence of signals at δc 138.1 and 129.2 (Stigmasterol nucleus). The 13C-NMR data of the sugar moiety combined with the chromatographic examination of the hydrolytic products (TLC and PC), allowed the identification of β-D-glucose as the sugar component (co-chromatography) in both compounds S2 and S3.
From the above data compound S2 was identified as β-sitosterol-3-O-β-D-glucoside while compound S3 was identified as Stigmasterol 3-O-β-D-glucoside.
Constituents of the Ethyl acetate and n-Butanol Fractions
Compound S4: Yellow powder, 50 mg, (fraction Fe3), m.p
313-314 oC, RF 0.6, (System: chloroform-methanol 8.5:1.5;
yellow in VL, yellow fluorescence in UV / NH3 or ALCl3).
Compound S5: Yellow powder, 30 mg, (fraction Fe3), m.p 330 oC, (Rf 0.5 System: chloroform-methanol 8.5:1.5; faint brown in VL, brown fluorescence in UV turning to yellow on exposure to NH3 and acquiring a yellow color when sprayed with AlCl3 yielding a yellow fluorescence in UV).
Compound S6: Yellow powder, 80 mg, (fraction Fe2),( Rf 0.37, System: chloroform-methanol 8.5:1.5; faint brown in VL, brown fluorescence in UV turning to yellow on exposure to NH3 and acquiring a yellow color when sprayed with AlCl3 yielding a yellow fluorescence in UV).
Compound S7: Yellow powder, 100 mg, (fraction Fb2), (Rf 0.45, chloroform-methanol-water 8:2:0.1; faint brown in VL, brown fluorescence in UV turning to yellow on exposure to NH3 and acquiring a yellow color when sprayed with AlCl3 yielding a yellow fluorescence in UV).
Compound S4 was identified as the flavonol aglycone quercetin based on co-chromatography alongside with an available authentic sample, m.p., m.m.p. and UV spectral data in methanol with or without the addition of different shift reagents as well as by comparison with reported data (Danial and Carl, 1969).
Compounds S5-S7: The UV analysis of compounds S5-S7 (Table 2) showed that they have most probably flavonol substituted skeleton (band I 328-358 nm). Bathochromic shifts in band I (34-55 nm) with increased intensity upon addition of sodium methoxide revealed the presence of a 4’ hydroxyl group and absence of free 3 or 7-hydroxyl group (no decomposition after 5 min). Which also proved by the bathochromic shift in band I upon addition of sodium acetate (13-24 nm).
In 1H- NMR of compounds S 5-S7 (table 3), the occurrence of two doublets signals at δH (6.40-6.63) and (6.67-6.76) (J = 2Hz ), indicated the presence of two meta protons at C-6 and C-8 of ring A, respectively. Signals at δH (7.35-8.0) (1H, d, J = 2.0 Hz ); δH (6.74-6.88) (1 H, d, J = 8.4 Hz ) and at δH (7.00-7.46) (dd, J = 2.0 and 8.4 Hz) indicated a 1’, 3’, 4’ tri-substituted system in ring B. In addition, compound S5 showed signal at 3.75 (3H, s, OCH3).
From the previous data and from the results of acid hydrolysis, isorhamnetin was identified as the aglycone moiety of compound S5 while was identified as the aglycone moiety of compounds S6 and S7.
The sugar part of compound S5 was identified as rhamnose by TLC after acid hydrolysis and by presence of an anomeric proton signal at δH 4.8 ppm (1 H br.s) and a methyl group at 1.2 ppm (3H,d, J = 2 Hz) .Comparison of 13C-NMR spectral data of the sugar moiety (102.7, 72.2, 75.4, 73.8, 73.5 and 18.6) with those published data, further confirmed the sugar moiety as rhamnose (Agrawal, 1989) (Table 4).
| Table 2: | Chromatographic and UV spectral data of compounds S4-S7 isolated from ethyl acetate and butanol extracts of Fagonia indica Burm F. |
| *Solvent system, Chloroform- methanol 8.5:1.5; ** Solvent system, Chloroform-methanol- water 8.0:2.0:0.1 | |
| Table 3: | 1H-NMR data (DMSO, 500 MHz, δ ppm) of compounds S5- S7 isolated from ethyl acetate and n- butanol extracts of Fagonia indica Burm F. |
From the above data, compound S5 could be identified as Isorhamnetin-α-3-O rhamnoside. In compound S6, the presence of 2 anomeric proton signals at δH 5.40 and 5.53 with J = 8, indicated the presence of 2 sugar units β-linked to the aglycone at C-3. The 13C-NMR data of the 2 sugar units (100.8,74.2,76.4,69.9,76.4 and 60.6 ;101,74.2,76.4,69.9,76.4 and 60.8) were in good agreement with those of glucose (Agrawal,1989). From the above data, compound S6 could be identified as Quercetin-3-O-β-D-glucopyranosyl-(1-6)-β-D glucopyranoside.
| Table 4: | 13C-NMR data (125 MHz, δ ppm, DMSO) of compounds S5- S7 isolated from ethyl acetate and n-butanol extracts of Fagonia indica Burm F. |
Compound S7 showed 3 anomeric proton signals indicated the presence of 3 sugar units. 2 of them appeared at δH 6.0 and 5.3 with J = 8 indicated the presence of 2 sugar units β-linked to the aglycone at C-3. The other sugar unit showed signals at δH 5.0 with J = 2, 1.2 (3H, d, J = 2 Hz) and at 1. 8 (3H,S) indicated the presence of 2'' O-acetyl rhamnose unit (Danial and Carl, 1969; Agrawal,1989). The 13C-NMR data of the 3 sugar units (104.9, 73.0, 73.3, 71.1, 74.5, 69.6; 101.8, 73.3, 79.3, 75.7, 69.6, 18.9, 27 and 105.0, 74.8, 78.2, 72.4, 77.6, 60.0) were in good agreement with those of galactopyranosyl -(6''-1''')- α-L-2''' acetyl rhamnose -(3'''-1'''') β-D-glucopyranoside (Agrawal,1989). From the above data, compound S7 could be identified as quercetin 3-O-β-D-galactopyranosyl -(6''-1''')- α-L-2''' acetyl rhamnose-(3'''-1'''') β-D-glucopyranoside which is a new compound isolated from Genus Fagonia. Structures are shown in Fig. 1.
| Fig. 1: | Structure of compounds S4, S6 and S7 |
| Fig. 2: | Effect of i.p injection of either aspirin (200 mg kg-1), or increasing doses of Fagonia indica Burm F. alcoholic extract on number of writhes per 10 min. Each bar represents the mean±SEM of 7 animals. *Significantly different from control group at p<0.05,@ significantly different from aspirin-treated group at p<0.05 |
Biological study
Acute toxicity testing of the total plant alcoholic extract: Oral
and intraperitoneal administration of the extract in doses up to about 4 g kg-1
body weight produced no mortality and no signs of morbidity or behavioral changes
in any of the animals of the treated groups during the period of observation.
This result indicates wide safety margin of the standardized Fagonia indica
Burm F. alcoholic extract and justifies its wide-spread use in folk medicine.
Antinociceptive activity of the total plant alcoholic extract
Writhing test: Results shown in Fig. 2 reveal that
the number of writhes induced by 0.6% acetic acid in normal mice reached an
average of about 42 writhes per 10 min. Aspirin produced about 80% inhibition
in the number of writhes induced by acetic acid in mice. Compared to aspirin,
2 mg of the Fagonia extract did not show any significant inhibition of
writhing response in the treated animals. On the other hand, 5 mg as well as
10 mg of the extract showed significant inhibition of the acetic acid-induced
writhing in mice. Both concentrations produced 52% inhibition of the writhing
response as compared to the control non-treated animals. Higher doses of the
extract (25, 50 and 100 mg kg-1) showed almost complete inhibition
of writhing indicating a maximum response.
Hot plate test: Results are demonstrated in Fig. 3 and 4. The latency period was found to be significantly increased upon treatment with morphine as compared to the normal control group which is in accordance with previous findings (Kim et al., 2005).
| Fig. 3: | Effect of i.p., injection of either morphine (10 mg kg-1), or increasing doses of Fagonia indica Burm F. alcoholic extract on the latency period of the hot-plate test in seconds. Each bar represents the Mean±SEM of 8 animals. *Significantly different from normal at the corresponding time interval, @Significantly different from morphine at the corresponding time interval, #Significantly different from the corresponding group at zero time |
| Fig. 4: | Effect of i.p., injection in presence of naloxone of either morphine (10 mg kg-1), or increasing doses of Fagonia indica Burm F. alcoholic extract on the latency period of the hot-plate test in seconds. Each bar represents the Mean±SEM of 8 animals. *Significantly different from normal at the corresponding time interval, @Significantly different from morphine at the corresponding time interval. Morphine-treated as well as Fagonia-treated groups at doses of 5, 10 and 25 mg kg-1 were significantly different from their corresponding values at zero time but marks of significance are omitted from the graph for simplicity |
Fagonia indica Burm F. alcoholic extract again showed antinociceptive effects with the higher doses tested at 30 as well as 60 min following administration. A dose of 25 mg kg-1 of the plant extract showed an analgesic effect which was non-significant from morphine at 30 min interval. In the present study, pretreatment of animals with naloxone produced similar analgesic effects with no significant differences in all groups except morphine group which shows significant attenuation of the hot-plate response of animals.
The previous results demonstrated that the alcoholic extract of Fagonia indica Burm F. was able to induce an analgesic/antinociceptive effect in mice in the writhing test. In this test, both peripheral and central analgesics could be evaluated and many investigators recommend it as a simple screening method (Vogel and Vogel, 1997; Talarek and Fidecka, 2002). In addition, the writhing t est possesses high sensitivity and it makes possible to detect analgesic activity of non-steroidal anti-inflammatory drugs. The peripheral analgesic response is thought to involve local peritoneal receptors and acetic acid causes algesia by liberating endogenous substances that excite pain endings (Raj, 1996). Aspirin, as is well established, inhibits cyclo-oxygenase in peripheral tissues, thus interferes with the mechanism of transduction in primary afferent nociceptors (Fields, 1987). At doses starting 25 mg kg-1, the extract produced complete inhibition of writhing response which indicates maximum response and confirms a potent analgesic effect mediated peripherally. This effect is probably related to decreased sensitization of nociceptive receptors to prostaglandins since these substances are the main mediators of pain transmission through the peripheral neurons (Dickenson, 1995).
A dose-dependent antinociceptive effect in the acute thermal pain model (hot-plate test) was also verified in mice upon treatment with the alcoholic extract of Fagonia indica Burm F. A prolongation of the latency period was observed following pre-treatment with morphine as well as the higher two doses of the extract suggesting that the plant extract has possible central analgesic properties. Morphine, acts by combining to opioid receptors and this in turn acts by either increasing the membrane K+ conductance or decreasing Ca++ conductance (Khanna and Sharma, 2001). Ca++ channel blockers (Verma, 1997) and K+ channel openers (Malhotra et al., 1997) both have been shown to have analgesic action of their own and they potentiate the analgesic effect of morphine. In the present study, 2 mg kg-1 of naloxone co-administration to animals treated with the different doses of the plant extract produced no significant differences in analgesic activity. These results could indicate that the central endogenous opioid system is probably not involved in the antinociceptive action achieved by the plant extract. Noteworthy, it was reported in other studies that naloxone was used at 5 and 10 mg kg-1 (Jayaram et al., 1995; Sawynok et al., 1995) which raises the possibility of obtaining different results upon using doses of naloxone higher than that used in the present investigation. Could the extract possess any of the abovementioned antinociceptive pathways involving K+ or Ca++ conductance needs further studies to be investigated and confirmed.
The antinociceptive effect of Fagonia indica Burm F. demonstrated in this investigation could be attributed, at least in part, to the presence, among its constituents, of flavonoids especially quercetin and its glycosides. Indeed, other studies have demonstrated that various flavonoids such as rutin, quercetin, hesperidin and biflavonoids produced significant antinociceptive and/or anti-inflammatory activities (Galati et al., 1994; Ramesh et al., 1998).
Antimicrobial screening
Under the experimental conditions used and as shown in Table 5 the following could be concluded:
| • | All the extracts at dose of 200 mg mL-1 exhibit marked antimicrobial activity against gram negative bacteria (pseudomonas aerugenosa and E. coli) comparing with the standard antibiotics, except hexane extract showed no activity against pseudomonas aerugenosa |
| • | Hexane and butanol extracts only showed activity at dose of 200 mg mL-1 against candida albican |
| • | Almost all the extracts (except butanol extract) showed activity against proteus |
| • | All the extracts in a dose of 200 mg mL-1 showed no activity against Streptococcus beta heamolytica |
Anti-tumor activities of the total plant alcoholic extract, the ethyl acetate and the butanol fractions: In a continuation of our interest in the chemical composition and biological activities of the Fagonia indica Burm F. the antitumor activity of the alcoholic extract and their fractions (ethyl acetate and butanol) which contain mainly flavonoids constituents was reported.
Under the experimental conditions used and as shown in Table 6 the following could be concluded: all the tested extracts exhibited marked antitumor activity against Memory Carcinoma F7 (MCF7) and Human Colon tumor cells (HCT).
| Table 5: | Effect of the different extracts of Fagonia indica plant on selected bacteria and fungi |
| - :No. inhibition zone; ++ :Diameter of inhibition zone 12-15 mm; +++ :Diameter of inhibition zone 16-22 mm; ++++ :Diameter of inhibition zone more than 23 mm | |
| Table 6: | Effects of the total alcoholic extract of Fagonia indica Burm F. and its prepared ethyl acetate and butanol fractions on MCF7 and HCT |
The ethyl acetate fraction showed least IC50 against both carcinoma cells MCF7 and HCT cells (30.5 and 31.9 μg mL-1, respectively) followed by n-butanol fraction which showed IC50 45.3 μg mL-1 against MCF7 while the alcoholic extract showed IC50 36.9 μg mL-1 against HCT.
CONCLUSION
Fagonia indica Burm F. plant collected from UAE desert was found to contain a moderately high percentage (3%) of flavonoid. The alcoholic extract of the whole plant possesses analgesic action which is probably mediated through both central and peripheral mechanisms and does not seem to involve opioid receptors. Moreover, the antitumor and the antimicrobial activities of the alcoholic extract and its fractions were also documented. These results provide some pharmacological rationale for the traditional use of the plant as analgesic and antitumor in folk medicine.
ACKNOWLEDGMENT
The authors thank Prof. Dr. Samia Shoman, Biochemistry Department, National Cancer Institute, Cairo, Egypt for carrying the antitumor activity of Fagonia extract.
A special acknowledgement to the fourth year students of the research groups of Pharmacognosy and Pharmacology Departments, Academic year (2008-2009), for their interesting attention and assistance of the research done in DPC.