ZANCO Journal of Pure and Applied Sciences The official scientific journal of Salahaddin University-Erbil https://zancojournals.su.edu.krd/index.php/JPAS
ISSN (print ):2218-0230, ISSN (online): 2412-3986, DOI: http://dx.doi.org/10.21271/zjpas
RESEARCH PAPER
Phytochemical study of Asplenium ceterach L. (Aspleniaceae) from Erbil province, Kurdistan of Iraq Ismail, A.M.1, Hamdi, B.A2, Maulood, B.K.3 & Al-Khasrejy, T.O.4 &Maythem AL-Amery1 1-Biology Department, College of Science for Women, University of Baghdad. Baghdad
2- College of Pharmacy, Hawler Medical University- Erbil 3- Biology Department, College of Science, Salahaddin University- Erbil, Kurdistan. 4-Biology Department, College of Education for Pure Science, University of Tikrit.
A B S T R A C T Asplenium ceterach L. (Rustyback fern) is a plant species traditionally used in Mediterranean countries and a traditional remedy for several diseases such as kidney stone and spleen complaints. In the current study a phytochemical analysis of the sporophytes focused on the constituents of Asplenium ceterach L. (Rustyback fern). Mature sporophyte of the fern was collected from Malakan in Erbil district during May – July 2016. As the phytochemicals qualitatively was screened had showed three flavonoid compounds: keampferol, keampferol- 3-O- glycoside and leutolin. Kaempferol was the dominant compound 130 µg/ml, while the total flavonoids concentration was only 265 µg/ml.in addition the screening shows that alkaloids, tannins, flavonoids and saponin were presented.
KEY WORDS: Phytochemicals Aspleniaceae Rustyback ferm.. DOI: http://dx.doi.org/10.21271/ZJPAS.33.s1.20 ZJPAS (2021) , 33(s1);187-191.
1. INTRODUCTION Works and investigations have been carried out on the phytochemical composition of ferns It is a quite evident from most recent studies on even thought their ethno botanical classification of ferns that Polypodiopsida importance also has been performed by includes only 7 orders, 33 families, and 300 numerous of genera with only 8861 species (Smith et al, 2006). The family Aspleniaceae which may be authors (Benniamin,2011; DE Britto et al, regarded as one of the most divers family 2012) . These specific role in habitat includes over 700 species, which involve conservation (Soare, 2008). terrestrial, epiphytic, epillithic species sub The fern Asplenium ceterach L., Family: Aspleniaceae, cosmopolitan. Its distribution and properties in order polypodiales, is widely spread species, small tufted wall fern with dark green, perennial fronds, up to 20 cm tropics can be recognized by ascending or sub long, penalty divided and incrusted on the underside with erect rhizomes, (Smith et al, 2006). rusty red-brown scales; these hide the sporangia, which lie along the veins. It was being used medicinally as a diuretic,
* Corresponding Author: Ismail A. M. E-mail: [email protected] Article History: Published: 15/03 /2021
ZANCO Journal of Pure and Applied Sciences 2021 Ismail, A.M..et al /ZJPAS: 2021, 33 (s1): 187-191 187
expectorant, against spleen complaints, kidney stones and hemorrhoids (Guarrera & Lucia, 2007). Bioactive compounds of ferns mainly belong phenolic, flavonoids, alkaloids, and terpenoids families (Ho et al, 2011), and Asplenium species mainly sporophytes, have been only partially phytochemicaly characterized, (Durdavic et al, 2007; Mir et al, 2013). The present investigation concern of the methanol extract of flavonoid content of Asplenium ceterach within Iraq. For best of our knowledge, no report so far available in the Figure 1. Asplenium ceterach L. collected from literature for the phytochemical screening in Malakan in Erbil district during May – July 2016 the region of this plant species. Therefor the 2.2. Plant raw materials present investigation may be regarded as the first study aimed to estimate the chemical Mature sporophyte of A. ceterach content of this species in Iraq, which may be (rustyback fern) were collected from Malakan used and practiced in medicinal ferns. region, plant spaceman was classified in accordance to (Ismail, 2018). Voucher fern sample with a number (4,17,1, As, ce) was 2. MATERIALS AND METHODS deposited in the herbarium at the Howler Botanical Garden – Erbil Governorate. 2.1. Study area Meteorological data (annual means) of the Malakan is a quite attractive region for study area recorded from metrological station, tourism that is situated along head of Erbil Governorate and presented in Table 1. Khlaifan river 74 km North West of Erbil city, Table 1- Metrological characters of the study area. it is located at 558 m above sea level at latitude 44° 26 533 E and longitude 36° 37 Region Temperature Rainfall Humidity 496 N, a water channel passes through (C°) (mm) (%) beautiful mountains region weed forests, and hedge banks, the climate is cold in winter, Malakan 39 120 59 snowfall begin from October to April, the rocks of hills and mountains are of calcareous type, (Townsend& Guest, 1966; Aziz, 2017). Generally, ferns were confined to this area in 2.3. Preparation of methanol extration the form of tufted winter green fern, the plants are so distinct in their general Plant material had been crushed and appearance that only little chance of powdered using electrical grinder then confusion may arise. Plant species have been extracted with 99% methanol (BDH), in collected throughout 2016 during May-July shaker water bath at 40 c° for 12 hour, then from different habitats all over the area (figure 1).
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after centrifugation for 10 min at 10000 rpm, filtered. One ml of the filtrate plant extract the supernatant were filtered with Millipore mixed with 5% FeCl3 (1 ml), appearance of filter (pore size 0.45µm) and further dried in brownish color indicates the presence of vacuum desiccator and then stored at 4 c° for tannins. further analysis. 3. QUANTITATIVE ANALYSIS 2.4. Phytochemical screening High Performance Liquid Chromatography Qualitative analysis of the plant crud extract (HPLC) technique was used for phytochemical cured out in advanced Biotech. Laboratory of analysis quantitatively following (Suarez et al Baghdad University – College of science, 2005). following (Harborne, 1998). The standard flavonoids injected in HPLC a- Flavonoids test instrument by an auto sampler according to optimum condition. The plant extract (20 µl) One ml of plant extract was mixed with (4 was injected in the HPLC instrument, the area ml) of 1N NaOH in a test tube, formation of a and retention time were recorded and then dark color was observed indicates the presence compared with the standard compounds Table 2 of flavonoids. & figure 2. b- Alkaloids test Column: C18-DB (50X 2 mm ID) Mobile phase: One ml of plant extract added to (2 ml) A= 0.05% Trifloro acetic acid (TFA) Myers reagent, a turbidity green color was observed indicates the presence of alkaloids. B= 0.1% Formic acid+ Deionized water+ Citric acetic c- Saponins test Flow rate: 1.1ml/min. Plant extract (0.5 gm) dissolved in (5 ml) Detection: UV at 280 nm. boiling distilled water in a test tube allowed to Calculation: conc. of sample µg/ml= area of cool and shake well then mixed thoroughly, the sample/area of stand. X conc. of stand x appearance of foam indicates the presence of dilution factor saponins. Standard solution concentration: 25 µg/ml. d- Tannins test Plant extract (0.5 gm) was dissolved in (10 ml) boiled distilled water in a test tube and then
Table 2- The area and retention time of standard flavonoids.
No. Compounds Retention time (min) Area
1 Leutoline 3.47 114892
2 Kaempferol 4.37 109560
3 Kaempferol-3-O-glycosid 5.29 107439
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Figure 2. HPLC chromatogram for flavonoids in table 2.the figure cited from the thesis belongs to the first author, for this other retention times were occurred.
4. RESULTS AND DISCUSSION compounds alkaloids, saponins, tannins and flavonoids, Table 3. 4.1. Phytochemical screening The methanolic plant extract was gave positive reactions for the presence of bioactive
Table 3- Phytochemicals methanolic extract of A. cetarach.
Plant Alkaloids Flavonoids Tannins Saponins
A. cetarach + + + +
4.2 Quantitative analysis Through HPLC analysis identified three species. The scientific researches were showed flavonoids were identified: kaempferol, that the ethanol and methanol extracts of keampferol-3-O- glycoside and leutolin. different species of Asplenium have a good Keampferol was shown to be the most antioxidant activity (Iwashina & Matsumato, dominant flavonoid as it recorded (130µg/ml), 2011; Pan et al, 2011; Nath et al, 2013). The while kaempferol-3-O- glycoside showed least investigations about in comparison and lowest concentration (31.7 µg/ ml), Table photochemistry of A. ceterach is quit rear 4. Previous studies on some species of comparison with other species of Asplenium , Asplenium revealed that the Asplenium is a therefore the present study regarded as the base rich source of alkaloids, saponins, and line studies and will give well information for flavonoids (Iwashina & Matsumato, 2000; future researches especially the uses of Ondo et al, 2015). So Mizano et al (1990) medicinal ferns in Iraq and the region. The reported that the flavonoids regard important results of quantitative analysis in plant extract active compounds to recognize Asplenium presented in Table 4.
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Table 4- Concentrations of flavonoids (µg/ ml) in A. ceterach extract.
Plant Quercetin Rutin Kaempferol Kaempferol_3_o Luteolin Myracetin Total _ Flavonoids glycoside A. ------130 31.7 103.3 ------265 ceterach
*Absent ------CONCLUSION
The results from the current study shows that mature sporophyte of the fern that collected from Malakan in Erbil district had showed
three flavonoid compounds: keampferol, keampferol-3-O-glycoside and leutolin. Kaempferol was the dominant compound 130 µg/ml,
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