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2290 J. Med. Plants Res. used as febrifuge, as well as to treat coughs and tuberculosis (Burkill, 1985). A decoction of the aerial part is used in Northern Nigeria as an antimalarial. In view of the enormous bioactivity profile and the documented ethnomedicinal uses of C. ambrosioides, this study aims to establish its phytochemical, pharmacognostic and thin layer chromatographic characteristics that could be useful for standardization and monograph development of this potential drug plant. MATERIALS AND METHODS All chemicals used were of analytical reagent grade (Sigma) and used as supplied. Plant materials and processing Plant material was collected by Mallam Muazzam Ibrahim from Chaza in Suleja, Niger state, Nigeria. The plant was identified and authenticated at the Herbarium of the National Institute for Pharmaceutical Research and Development, Abuja, Nigeria. The aerial part of the plant was air-dried for three weeks at room temperature, pulverized and used immediately for analyses. Phytochemical screening The phytochemical and proximate analyses of the powdered aerial part of the plant were performed using the methods described by Harborne (1998), MHFW (1999), Evans (2002) and Sofowora (2008). The analyses were carried out as thus explained. Test for cardenolide aglycone 0.5 g of the powdered sample was boiled with 10 ml of 95% alcohol for 2 min. The mixture was filtered; the filtrate was allowed to cool and diluted with water to 10 ml. Three drops of a saturated solution of lead sub-acetate were added and filtered thoroughly. The filtrate was treated with 1 ml of 2% solution of 3, 5-dintrobenzoic acid in 95% alcohol and the solution basified with 5% sodium hydroxide. A purple-blue colour indicates the presence of free or combined cardenolide aglycone (Sofowora, 2008). Test for terpenes and sterols 5 g of the powdered sample was extracted by maceration with 50 ml of chloroform. The extract was filtered and evaporated to dryness on water bath. The residue was dissolved in 10 ml of anhydrous chloroform and filtered. The filtrate was divided into two equal portions and used for the following tests: Lieberman-Burchard test for terpenes: To the first portion of the chloroform solution was added to 1 ml of acetic anhydride and shaken. Then 1 ml of concentrated sulphuric acid was added down the wall of the test tube to form a layer underneath. The formation of a reddish-violet colour at the lower layer indicates the presence of terpenes (Sofowora, 2008). Salkowski’s test for sterols: The second portion of the chloroform solution was carefully mixed with 2 ml of concentrated sulphuric acid so that the sulphuric acid formed a lower layer. A reddish- brown colour at the interface indicates the presence of a steroidal ring. Test for saponins 0.5 g of the powdered plant material was added to 5 ml of 95% ethanol and boiled for 2 min; filtered into a test tube and diluted to 10 ml with distilled water. The test tube was shaken vigorously for 1 min. The formation of a persisting honey comb indicates the presence of saponins. Test for tannins and phlobatannins Ferric chloride test for tannins: 10 ml of distilled water was added to 1 g of the powdered sample in a test tube and boiled for 3 min in a water bath. The mixture was allowed to cool and then filtered with Whatman No.1 filter paper. 1 ml of the filtrate was diluted with 4 ml of distilled water and few drops of 10% ferric chloride were added. Instant formation of blue-black or green coloured solution indicates the presence of tannins (Evans, 2002). Test for Phlobatannins: 10 ml of distilled water was added to 1g of the powdered sample in a test tube and boiled for 3 min in a water bath. The mixture was allowed to cool and then filtered with Whatman No. 1 filter paper. 1 ml of the filtrate was boiled with equal volume of 1% v/v hydrochloric acid. The formation of a red precipitate indicates the presence of phlobatannins (Evans, 2002). Test for anthraquinone (Borntrager’s tests) 0.5 g of the powdered sample was poured into a dried test tube and 10 ml of chloroform added. The mixture was shaken for 5 min and filtered. The filtrate was shaken with 10% w/v potassium hydroxide solution. A bright pink-red colour in the upper aqueous layer indicates the presence of free anthraquinones (Evans, 2002). Test for balsams 1 g of the powdered sample in a test tube was extracted with 10 ml of 90% v/v ethanol and filtered. Two drops of 10% (w/v) alcoholic ferric chloride solution was added to 5 ml 5 of the filtrate. Formation of a dark green coloured solution indicates the presence of balsams. Tests for resins 0.5 g of the pulverized plant was extracted with 15 ml petroleum ether and filtered. 5 ml of the filtrate was dispensed into a test tube and shaken vigorously with equal volume of copper acetate solution TS. The mixture was allowed to stand for a few minutes. Formation of a green coloured solution indicates the presence of resins. Test for alkaloids 3 g of the powdered sample was macerated with 50 ml of methanol at room temperature and filtrate evaporated to dryness on a hot water bath without overheating. 10 ml of 1% v/v HCL was added to the residue. The solution was divided equally into five test tubes, and two drops of the following reagents were added to the respective test tubes: Mayer’s reagent – (potassium mercuric iodide
Table 1. Result of phytochemical screening of the aerial part of C. ambrosioides. Phytochemical constituents Result Cardenolide aglycone + Terpenes + Sterols + Saponins + Tannins + Anthraquinones ND Balsams ND Resins ND Alkaloids + Phlobatannins ND Flavonoids + Phenols + Volatile oil + +: Detected; ND: not detected. solution); Dragendorff’s reagent - (potassium bismuth iodide solution); Wagner’s reagent – (solution of iodine in potassium iodide); Hager’s reagent – (a saturated solution of picric acid); and 10% tannic acid solution. The formation of amorphous or crystalline precipitates or coloured precipitate in at least 3 or all of these tests indicates the presence of alkaloids. Test for flavonoids Lead acetate test: 5 g of the powdered sample was detanned by wetting it with acetone, and the acetone was completely evaporated on a hot water bath. The residue was extracted with 20 ml of warm distilled water and filtered. 5 ml of the filtrate in a test tube was added two drops of 10% (w/v) lead acetate solution. Formation of a coloured precipitate indicates the presence of flavonoids. Sodium hydroxide test: To 5 ml of the filtrate from above equal volume of 10% (w/v) sodium hydroxide solution was added. Formation of yellow coloured solution indicates the presence of flavonoids. Test for volatile oil 0.5 g of powdered sample was shaken with 1 ml of 0.1 M sodium hydroxide solution and 1% aqueous hydrochloric acid. The formation of a white precipitate indicates the presence of volatile oil. Determination of water-soluble ash Water soluble ash was determined as reported in MHFW (1999), with slight modification. Briefly, total ash was determined. The total ash was boiled for 5 min with 25 ml of distilled water; the insoluble matter was collect on an ashless filter paper, washed with hot distilled water, and ignited for 15 minutes at a temperature not exceeding 450°C. The weight of the insoluble matter was subtracted from the weight of the total ash; the difference in weight represents the water-soluble ash. The percentage of the watersoluble ash was calculated with reference to the air-dried powdered plant sample. Okhale et al. 2291 Extraction and thin layer chromatographic fingerprinting Successive extraction of 1 g powdered aerial part was carried out with n-hexane (10 ml × 2); ethyl acetate (10 ml × 2); and methanol (10 ml × 2) at room temperature (27 to 30°C) for 24 h. The extract from each solvent was vacuum filtered with Whatman No. 1 filter paper, and the filtrates evaporated to dryness in the fume hood at room temperature. The yield for each extract was determined. Then 0.05 g each of the extracts was reconstituted in 5 ml of their respective solvent of extraction and spotted on glass TLC plate precoated with silica gel 60. The plate was previously activated at 105°C for 2 h. The plates were developed using a mobile phase comprising n-hexane and ethyl acetate (3: 2). The plates were observed in daylight and under UV at 365 nm. Visible spot were marked. The plates were then sprayed with a solution of 1% (w/v) vanillin in sulphuric acid and heated in oven at 110°C for 3 min. The coloured spots revealed after spraying were marked. The retardation factors (Rf) of all components detected were then computed. RESULTS AND DISCUSSION Phytochemical screening The results of the phytochemical screening of the aerial part of Chenopodium ambrosioides collected from Northern Nigeria are presented in Table 1. Nine major classes of secondary metabolites were detected, namely cardenolide aglycone, terpenes, sterols, saponins, tannins, alkaloids, flavonoids, phenols and volatile oil. C. ambrosioides has been shown to exhibit antifungal, antihelminthic, anticatarrhal, antibacterial, antiviral, insecticidal, nematicidal and allelopathic activities (Verma et al., 1983; Dubeyand Kishore, 1987; Peterson et al., 1989; Begum et al., 1993; Kishore et al., 1993; Hegazy and Farrag, 2007; Valery et al., 2008). The antihelminthic and anticatarrhal activities have been attributed to the presence of some bioactive compounds shown in Figure 1 such as ascaridole (1), isoascaridole (2), α-terpinene (3) and ascaridole glycol (4) (Valery et al., 2008). Ascaridole has been reported to possess sedative, painrelieving and antifungal properties (Okuyama et al., 1993; Pare et al., 1993). It has also been reported to exhibit antimalarial and antiparasitic activities. Pollack et al. (1990) reported its inhibition of the in vitro development of Plasmodium falciparum, while its activities against Trypanosoma cruzi and Leishmania amazonensis were reported by Kiuchi et al. (2002) and Monzote et al. (2006), respectively. Ascaridole was also reported to exhibit in vitro activity against different tumor cell lines (CCRF-CEM, HL60, and MDA-MB-231) (Valery et al., 2008). Our finding is in agreement with previously reported work of Hegazy and Farrag, (2007), who reported the presence of sterols and terpenes in the Egyptian species, and Onocha et al. (1999), who reported the presence of essential oil in Chenopodium ambrosioides collected from Western Nigeria. Several other secondary metabolites have been previously isolated from Chenopodium
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Journal of Medicinal Plants Researc
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Editors Prof. Akah Peter Achunike E
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Electronic submission of manuscript
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Fees and Charges: Authors are requi
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Table of Contents: Volume 6 Number
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et al., 1996; van Wyk and Gericke,
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DPPH inhibition (%) DPPH % inhibiti
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% Mortality Concentration (µg/ml)
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information on plants used for the
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Table 1. Levels of independent vari
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Arbutin (mg/g) Co-solvent Figure 1.
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Arbutin (mg/g) Extraction pressure
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Table 3. Arbuitn content of Asian p
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Arbutin (mg/g) Extraction pressure
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Arbutin (mg/g) Extraction temperatu
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Table 1. Chemical composition of es
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Darjazi 2369 Table 2. Statistical a
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Table 3. Correlation matrix (number
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Table 1. Precision test. extraction
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Figure 2. Effect of different alcoh
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Table 6. Results of ANOVA. Nie et a
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Table 2. Plant data and MIC values
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exhibiting the polar extracts of P.
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Table 3. Contd. AcOEt ++ ++ ++ - Me
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Figure 1. Map of Ladakh region of I
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Warghat et al. 2391 Table 3. Summar
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Table 5. Inter-Population genetic i
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Excoffier L, Smouse PE, Quattro JM
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ice residue is an ideal raw materia
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DPPH• scavenging ratio(%) OD valu
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scavenging effect on free radical a
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our new CL system. Fenton reaction
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1 2 Time (s) Figure 2. Kinetic curv
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Wong et al., 2006). It can be seen
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Pan YM, Liang Y, Wang HS, Liang M (
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2422 J. Med. Plants Res. close cano
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2440 J. Med. Plants Res. Table 1. R
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2442 J. Med. Plants Res. Stability
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2444 J. Med. Plants Res. production
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2486 J. Med. Plants Res. components
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UPCOMING CONFERENCES 15th European
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