MORPHOLOGICAL AND CHEMICAL CHARACTERISTICS OF TWO SPECIES BELONG TO ALYSSEAE AND LEPIDIEAE TRIBES SPREAD IN NORTHERN IRAQ

This research was aimed to study three species which are prevalent in northern Iraq: Alyssium strigosum Banks and Sol., Clypeola jonthlaspi L, and Isatis tinctoria L. belonging to the Alysseae and Lepidieae tribes. The general characteristics of the roots, stems, leaves, fruits and seeds are studied and it turned out that and the two species A. strigosum and C. jonthlaspi are similar due to their belonging to the Alysseae tribe, and the species I. tinctoria differs since it belongs to the Lepidieae tribe. In addition, 6 secondary metabolites are diagnosed using the qualitative tests: alkalis, phenols, tannins, flavonoids, glycosides, and sapindales. The presence of terpenoids was not observed, and the alcohol extract is superior to the aqueous extract regarding the accuracy of the results. The phenols are detected using HPLC technology and four compounds are found: Rutin, Quercetin, Kaempferol and P-Coumarin. The importance of studying the chemical content comes from its use in subsequent studies and knowledge of its uses in the medical fields.


INTRODUCTION
The mustard family (Brassicaceae or Crucifera) is considered as the fourth largest flower family. It is easily diagnosed based on the actinomorphic flowers, the Cruciform corolla, and the type of fruit whether Silicula or Silique (7,9) . The Brassicaceae includes about 338 -380 genera and 3000-4060 species, which are spread globally in the temperate regions of the northern hemisphere and are found worldwide ( 6,11,16). In Iraq, this family includes 5 tribes, 80 genera, and 177 species (10,20,39). In the Turkish Botanical Encyclopedia, it has 91 genera, 555 species, 51 subspecies, 22 varieties and 621 orders (13,14,17). Also, there are 120 genera and 358 species in Iran (22). The significance of the Brassicaceae is that it is one of the 10 most important families in economic terms, and includes many economic crops (10). There are many studies focus on the exact morphological traits, including Orcan and Binzet (28). study of Alyssum obtusifolium species (21). Also studied the exact morphological properties of Clypeola L. genus pollen in Iran, (33) studied the morphological and anatomical features of stems and leaves for the Ricotia L. genus grown in Turkey. Whereas Abbasian & Keshavarzi (1) studied the morphological and exact morphological traits of the Clypeola genus in Iran. Al-abide and Al-Shamary(4) study the indumentum ,crystals and stomata in 14 species from the tribe Brassiceae for the Brassicaceae in Iraq, as well as Al-abide (7) studied the morphological properties of four species of the Lepidieae tribe: Aethionema cordifolium DC., Biscutella ciliate L., Thlaspi perfoliatum L., and Calepina irregularis Asso. that spread in Iraq, specifically in Erbil Governorate. In addition, a taxonomic, morphological and anatomical study has been conducted on the reproductive parts (fruits and seeds) for different species of the Brassicaceae in Iraq by (5). There are also few chemical studies of some different types, including the study by Al-abide et al. (8)and the study of Obeid and Jaber (27) of Pelargonium graveolens species. which focuses on discovering the effective chemical compounds of some species of the Brassicaceae, its biological efficacy and the effect on the growth of some Candida fungi. Also, a study by Saeed (31) of four species related to the Arabis L. genus and the diagnosis of some phenolic compounds. The current research aims to collect the largest amount of information concerning the morphological differences and classification of the studied species using chemical evidence in order to facilitate their field diagnosis in addition to knowing and diagnosing the effective chemical compounds of wild plants and the extent of their future use as an alternative to the manufactured chemical compounds.

MATERIALS AND METHODS Specimens' collection
The plant samples of the Brassicaceae are collected through several field trips to northern Iraq, specifically in the mountainous areas of Erbil Governorate, in the spring season for the three years between 2018-2020. The collected samples are diagnosed based on the available botanical encyclopedias (Iraqi, Turkish, Iranian and Saudi Arabian (13,23,37) and compared with the dry samples deposited in the Iraqi National Herbarium at Abu Ghraib. The morphologically fresh samples are studied and differences are determined for the fresh parts represented by roots, stems and leaves using the scaled ruler. The reproductive parts (flowers, fruits and seeds) are also studied under an anatomical microscope and by using the scaled ophthalmic lens under the powers 4x, 10x and 40x, measurements are taken and tabulated in tables and photographed using the Nikon camera using the method of Alabide (7). Preparation of aqueous and alcoholic extracts: The hot aqueous extract is prepared by mixing 5 gm of plant sample powder (Aerial parts) with 25ml of hot distilled water, put into the vibrator for 10 minutes, then left for 24 hours. (30)The mixture is filtered through Whatman No.1 filter paper, and the filtrate is collected and put in opaque bottles and kept in the refrigerator until the qualitative (inferential) checks are made. The same previous method is adopted and the water is replaced with ethyl alcohol with a concentration of only 96%, after which several inductive tests are performed to detect the presence of alkaloids using Mayer's and Wagner's test, phenols, flavonoids, glycosides, sapindale, terpenoids and tannins by following the method mentioned in (2,30,38). Extraction 10 gm of plant powder were dissolved in 200 ml hexane to remove fat, resin, then 200 ml of 80:20 ( methanol: water ), The extract was subjected to ultra-sonication (Branson sonifier, USA) at 60% duty cycle for 25 min at 25C, followed by centrifugation at 7,500 rpm for 15 min . The clear supernatant of each sample was decantation and filtered through filter paper Watman no 1, then the aqueous extraction was evaporated under vacuum (Buchi Rowas evaporated Re type). Dried samples were re-suspended in 1.0ml HPLC grade methanol by vortexing, the mixture was passed through 2.5 mµ disposable filter, and stored at 4℃ for further analysis, then 20 µl of the sample injected into the HPLC system according to the optimum condition. (24 , 36).

Separation condition
The main compound was separated on FLC (Fast Liquid Chromatographic) column under the optimum condition . Column: phenomenex C-18,3µm particle size (50 X 2.0 mm I.D),Column Mobile phase:linear gradient of solvent A0.1% formic acid, gradient program form 0% B to 100% B for 10 minutes .Flow rate 1.2ml\ min . Detection :UV280 nm. The separation occurred on liquid chromatography Shimadzu 10AV-LC equipped with binary delivery pump model LC-10A Shimadzu, the eluted peaks were monitored by UV-Vis 10A-SPD spectrophotometer. Chemical compounds in the species under study were estimated based on (35). As shown in (Table 1)    The roots are tap roots of the conical type, not branching in A. strigosum, and C. jonthlaspi species. Except for the I. tinctoria species, the roots are wedge-branched at the base of the root. Its length ranged from 2-13 mm, with the lowest root length in A. strigosum species and highest in I. tinctoria species. (Table 2 and Plate 1). The roots are similar in all the studied species for the reason of their growth in the mountainous region, the lack of surface water and the nature of the rocky soil. Since the roots are affected by environmental factors faster than other plant parts, changes appear faster,whereas C. jonthlaspi and I. tinctoria were similar to the apical polymorphic erect stem, while the stem of A. strigosum is un branched (Plate 1), while the differences of the stems are due to the impact of this trait by many environmental and genetic factors. Examination of the leaves show that all are simple, undivided, and cordate in I. tinctoria and elongated in A. strigosum and C. jonthlaspi. The leaf apex varies in shape, with acute in I. tinctoria and obtuse in A. strigosum and C. jonthlaspi. Cordate base in I. tinctoria, truncate plane in A. strigosum, C. jonthlaspi and undulate margin in I. tinctoria and entire smoothness in the other species under study, while leaf size ranged between 10-40 mm and average width between 2-20 mm. (Table 2 and Plate 1). The results of the current study show the similarity of the flowers of all the species under study. They are bisexual with white petals in A. strigosum and C. jonthlaspi except for I. tinctoria which appears with yellow petals while the fruits are of the silicula type.
Here it should be noted that there is a difference in the shape of the fruits and they are ovate. Sides compressed in A. strigosum and C. jonthlaspi and oblong in I. tinctoria.
The size and shape of the fruit vary between 6-8 mm, the beak between 2-3 mm, and the petiolate size between 10-15 mm. The fruit contains 2 seeds and trichomes of the glandular and eglandular stellate cellmulticellular type in A. strigosum while the size of C. jonthlaspi fruit ranges from 4-6 mm. The beak ranges between 1-2 mm, the petiolate between 8-10 mm, its ovate shape containing a single seed, and the hairs covering of the glandular type. while the size of the beak ranges between 5-8 mm and the petiolate between 4-6 mm and fruit 15-20 mm which is smooth superficial containing a single seed in I. tinctoria. The results of the present study show a difference in seed shape among the species under study. They are oval in A. strigosum and C. jonthlaspi and oblong in I. tinctoria. In addition to the emergence of variation in seed color, they are dark brown in A. strigosum and I. tinctoria, and light brown in C. jonthlaspi, and the surface trimmings are smooth in C. jonthlaspi and I. tinctoria and granular in A. strigosum (Table 3 and Plate 2). The results of the present study are in agreement with the results of (3,18,24,25), which indicate the similarity of many appearance properties (shape, habitat, stem branching and leaf shape, apex, base and margin, fruit type and Indumentum surface of the seed) for both A. strigosum and C. jonthlaspi. The reason for this is the common origin of the two species and their affiliation to the same Alysseae tribe, while the different properties of I. tinctoria are due to the difference of its Isatideae tribe. De Candolle (15) and Al-Shehbaz et al. (10) classified the studied species and are placed within different families while Hayek (19) classified I. tinctoria within the Arabideae tribe, the subclan Isatidinae, while Schulz (32) placed it within Lepidieae subtribe Isatidinae. The difference in the dimensions and shapes of the fruits helps in the diagnosis of the studied species in the field.   1-A. strigosum 2-C. jonthlaspi 3-I. tinctoria .( A-plants, B-root , C-leaf) Alyssium strigosum 2.Clypeola jonthlaspi 3. Isatis tinctoria . ( A-fruit, B-seed).

Diagnosing chemical compounds:
The presence of alkaloids is detected in C. jonthlaspi and I. tinctoria and does not appear in A. strigosum using Mayer's and Wagner's test. The presence of flavonoids, phenols, glycoside glycosides, tannins, sapindales, and terpenoids are not detected in all studies. (Table 4 and Plate 3). The HPLC results show the diagnosis and the presence of four phenolic compounds in the species under study, the compounds are Rutin, Quercetin, Kaempferol and P-coumarin, and the concentrations of the compounds differ in different species. The highest concentration of rutin appears to be 377.3, 518.9 and 296. 3mg / ml in the species A. strigosum, C. jonthlaspi and I. tinctoria respectively. From the results above, it is found that the concentration of rutin is higher in the C. jonthlaspi, while the lowest concentration of quercetin is 97.1 mg / ml in the A. strigosum and reaches the lowest concentration of 246.4 mg / ml of p-coumarin reported in C. jonthlaspi, while the lowest concentration of kaempferol is 45.5 mg / ml in I. tinctoria (Figure 2,3 and Table 5). The results of Table .4 indicate the richness of the  studied plants with phenolic compounds,  flavonoids and glycosides. Tannins, Sapindales and lack of terpenoids. This indicates the exposure of plants to environmental stresses, which helps in building secondary metabolites and chemical compounds forming a defense system against bacteria, viruses ,fungi. and insects. In addition, the plant produces secondary metabolites as a means of confronting environmental stresses, and the diagnosis of these compounds may contribute to solving the classification problems through the use of indicators of Phytotaxonomy. From the results of Figuer 2,3 and Table .5 , it is evident that the alcoholic extract is superior in detecting compounds compared to the aqueous extract. The current study with the results of the (21). indicated the presence of many phenolic compounds within the cruciferous family plant. It also agrees with the results of Abbasian and Kishavarzi (1) in the detection of secondary metabolic compounds and their biological effectiveness against pathogens, as they state that the chemical compounds that are isolated from the leaves of the Brassicaceae using HPLC technology have an effective role against bacterial pathogens, as well as that phenolic compounds provide evidence that helps in classification of plants that contributes and enhances morphological properties (29), Shankar et al. (34) study indicates the use of Brassicaceae plants as main food plants for some of the world's population, and the oil extracted from their plants constitutes 14% of the vegetable oils suitable for human consumption. Its plants are an important source for medicinal, agricultural and economic purposes because they contain nutritional health features beneficial to the human body and possess antioxidants, including carotenoids, ascorbic acid and phenols that are used in the treatment of heart diseases and cancers (34). In addition, the concentration of phenolic compounds can be calculated to help choose the best types that can be used.Finally, it should be noted that the concentrations of phenolic compounds are important depending on many variables, such as the study method, environmental factors, time of collection, and place of collection (12). Therefore, this research mainly focuses on the morphological properties of some species of the Brassicaceae , in addition to the differences in the concentrations of phenolic compounds.    • The results showed a similarity in the morphological properties of the A. strigosum and C. jonthlaspi on the one hand (Apex, base and margin), while the I. tinctoria differed in its properties, and a difference appeared in the dimensions of leaves for the three studied species.
• It is found that the fruits of the two species A. strigosum and C. jonthlaspi are of the species silicle, while the fruit of the species I. tinctoria is of the type Silique.
• The emergence of a difference in the shapes and colors of the seeds in addition to the Indumentum dimensions and surface inscriptions of the three studied species. • Qualitative chemical tests indicate the presence of 6 secondary metabolites, which are alkaloids, phenols, tannins, Falvonoides, glycosides, sapindales, and the absence of terpenoids and the superiority of the alcoholic extract in detecting compounds compared to the aqueous extract. • HPLC analysis shows the presence of 4 phenolic compounds which are Rutin, Quercetin, Kaempferol and P-coumarin in all studied species and their concentration and ratios differ due to the difference in biological activity.

ACKNOWLEDGMENT
The author thanks the Department of Biology, College of Education for Pure Sciences, University of Tikrit, Iraq, for providing the facilities for the research work.=