Preliminary Phytochemical and Physicochemical Analysis of Selected Mistletoes from Ethiopia

Objectives: Mistletoes have benets for the treatment of human and animal health ailments. This study investigated the physicochemical and phytochemical constituents of Englerina woodfordioides, Phragmanthera regularis, Tapinanthus globiferus, and Viscum tuberculatum from central Ethiopia. Results: The four plant species collected from 11 host trees grown in six distinct locations revealed the presence of alkaloids, phenols, saponins, cardiac glycosides, steroids, terpenoids, tannins, quinines, and coumarins. In contrast, no avonoids, anthraquinones and phlobatannins were detected. The total ash value of E. woodfordiodes and P. regularis ranges from 6.04 to 13.23% and 5.62 to 15.22%, respectively. Comparable total ash content was found in T. globiferus and V. tuberculatum. However, no signicant difference was obtained in the mean percent moisture content of the study species.


Introduction
Mistletoes are hemi-parasitic parasitic owering plants found attached to the branches of other plants [1]. They are known as complementary and alternative medicine in the treatment and management of various diseases around the world [2][3][4][5]. The biological activities of mistletoe rely on the presence of secondary metabolites [6]. The phytochemical composition of African mistletoes is largely dependent on a few species of Tapinanthus and Phragmanthera species grown in West African countries. The phytochemical composition of these mistletoes varied depending on the time of harvest, host plants and the manufacturing process [7][8].
Ethiopia has long history and deep-rooted culture in using mistletoe to treat various health problems. Accordingly, Englerina woodfordiodes use to treat diarrhea and eye disease [9], syphilis [10], otorrhoea, and scabies [11]. Similarly, T. globiferus were reported in the treatment of spider poisoning, urinary problem, cough, blotting [12], and dyspepsia and impotence [13]. Despite these importances, to our knowledge, the study on the phytochemical composition of species of mistletoes achieved less attention in the past. Therefore, the aim of this study was to investigate the phytochemical and physicochemical properties of Phragmanthera regularis, Englerina woodfordioides, Tapinanthus globiferus and Viscum tuberculatum in Ethiopia.

Plant collection and authentication
Mistletoes were collected from branches of trees grown in the roadside, homesteads, and natural forests in six localities in Ethiopia in April 2019. These specimens were identi ed by Melaku Wondafrash, plant taxonomist, at the National Herbarium (ETH) of the College of Natural Sciences, Addis Ababa University. Specimens were deposited at ETH.

Preparation of plant crude extracts
Fresh and healthy leaves of mistletoes were washed several times with running tap water to remove soil and debris. Air-dried leaves were ne powdered using electrical grinder mill.
Extracts were kept in a glass jar for seven days with occasional shaking at room temperature. These extracts were ltered using Whatman no. 1 lter paper. The marc was re-macerated with the same solvent until the extraction was exhausted. The rst three solvents extracts were concentrated to dryness using a Rotary evaporator. The resulting aqueous ltrate was lyophilized.

Determination of solvent extraction yield
The crude extracting values of mistletoes were tested using the above-mentioned solvents. The percentage yield of extraction was determined as follows [14].

Determination of phytochemical constituents of plant extracts
Test for alkaloids Wagner's test crude extract powder was dissolved in de-ionized distilled water and ltered using Whatman no1 lter paper. The ltrate was acidi ed with hydrochloric acid (HCl) [15]. To this solution, Wagner's reagent was dissolved in water and gently added to the test tube [16][17]. The formation of reddish-brown precipitate indicates the presence of alkaloids [15].
Test for anthraquinones crude extract was dissolved in chloroform, shaken for 5 min [18], and ammonia solution was added. The solution was ltered using Whatman no 1 lter paper [19]. The control test was done by adding ammonia solution in chloroform [20]. The formation of bright pink coloration in the upper aqueous layer indicates the presence of anthraquinones [20].

Test for cardiac glycosides
Keller-Killani test crude extract was diluted in distilled water [21]. Two milliliters of plant extract [22] were mixed with glacial acetic acid followed by drop wise addition of ferric chloride (FeCl 3 ) solution. Then, the mixture was poured into another test tube containing concentrated sulfuric acid (H 2 SO 4 ). A brown ring formed at the interface indicates the presence of cardiac glycosides [21,23].

Test for coumarins
Sodium hydroxide (NaOH) test crude extract was dissolved in distilled water. Sodium hydroxide solution was added to the aqueous plant extract. The appearance of yellow color indicates the presence of coumarins [24][25].

Test for avonoids
NaOH or Alkaline Reagent test crude extract was dissolved in water and ltered using Whatman no 1 lter paper. The plant extract was treated with aqueous NaOH solution [22,25]. The formation of intense yellow color, which becomes colorless on the addition of dilute acid, indicates the presence of avonoids [23,[26][27][28].
Lead acetate test crude extract was dissolved in water and ltered using Whatman no 1 lter paper. To the extract, few drops of basic lead acetate solution were added. The formation of reddish-brown precipitate indicates the presence of avonoids [17]. FeCl 3 test crude extract was dissolved in water and ltered using Whatman no 1 lter paper. Few drops of neutral FeCl 3 solution were added to the extract. The deposition of blackish-red precipitate indicates the presence of avonoids [17].

Test for phenols
FeCl 3 test crude extract was mixed with distilled water [28]. The extract was treated with an aqueous FeCl 3 solution [29]. The formation of bluish-black color indicates the presence of phenols.

Test for phlobotannins
Precipitate test two milliliters of aqueous extract of the plant sample were boiled with HCl acid. The deposition of a red precipitate indicates the presence of phlobotannins [24][25].

Test for quinones
Two milliliters of plant extract were treated with HCl acid. The formation of a yellow-colored precipitate indicates the presence of quinines [30].

Test for saponins
Foam test plant extract was shaken vigorously with water. The appearance of foam indicates the presence of saponins [31][32].

Test for steroid
Liebermann-Burchard test a few drops of acetic anhydride solution were added to the extract. To this solution, a few drops of concentrated H 2 SO 4 were added carefully along the side of the test tube. Formation of reddish-brown ring at the junction of the two layers indicates the presence of steroids [17].
Test for tannins FeCl 3 test: plant extract was dissolved in distilled water, and then ltrated. Two milliliter FeCl 3 was added to the ltrate. The appearance of blue-black, greenish-black or dark blue color indicates the existence of tannins in the test samples [19].

Test for terpenoids
Salkowski's test plant extract was added to 2 ml of chloroform. Three milliliters of concentrated H 2 SO 4 were carefully added to form a layer. A reddish-brown coloration of the interface indicates the presence of terpenoids [21].
Test for reducing sugar Benedict test plant extract was dissolved in distilled water and ltered separately. One milliliter of ltrate and 4 ml of Benedict's reagent were mixed, and heated gently in a boiling water bath for 5 min. The appearance of green, red, or yellow coloration indicates the presence of reducing sugar in the plant extract [33].

Test for protein
Biuret test plant extract put in a test tube was treated with an equal volume of NaOH. Then, a few drops of CuSO4 were added. Pink or purple color indicates the presence of proteins [34].

Determination of physicochemical analysis of plant materials
Total ash values The total ash content of the plants was determined as stated in Idris et al [35] with some modi cations. A dry heat-resistant porcelain crucible was weight (W 1 ). Thereafter, 2 g of leaf powder was weighed along with crucible (W 2 ), and ignited gradually in an electrical mu e furnace, increasing the heat to 600 0 C until the plant materials were white that indicate the absence of carbon. The crucible was removed, and allowed to cool in a desiccator and then re-weighed (W 3 ).

Acid-insoluble and water-soluble ash
The acid-insoluble and water-soluble ashes were separately prepared by adding 25 ml of HCl into total ash content obtained in 2.6.1. The solutions were gently boiled for 5 min covered with a watch glass. The insoluble matters were ltered using Whatman no 40 Ashless lter paper. The ltrate was washed with hot water and then ignited for 15 minutes at 450 °C. The lter paper containing the insoluble matter were transferred to the original crucible, dried on a hot plate and ignited to 450 0 C to constant weight (W 4 ). The residue was cooled in desiccator and weighed.

Determination of moisture content
The moisture content (loss on drying) of the fresh leaves was determined as stated in Danso-Boateng et al [36]. The air-dried samples (3 g) were weighed and kept at 105 ± 3 0 C for 24 h. Samples were then removed from the oven, cooled in a desiccator, and weighed.

Plant collection and authentication
In this investigation, four mistletoes were collected from 11 host plants [ Table 1].

Phytochemical constituents of plant extracts
The phytochemical test showed that there were alkaloids, phenols, saponins, cardiac glycosides, steroids, terpenoids, tannins, quinines, and coumarins in one or the other four mistletoes. No avonoids, anthraquinone and phlobatannin were detected. E. woodfordioides and P. regularis have similar results in the contents of avonoids, cardiac glycosides, steroids, anthraquinones and phlobatanins. Tannins were found in all E. woodfordioides. Similar to this nding there was saponnins to all P. regularis collected from different host plants. In contrast, the phytochemical composition of V. tuberculatum was much similar to E. woodfordioides collected from E. globules than the others [ Table 2]. Meanwhile, a trace amount of reducing sugar was found in E. woodfordiodes, T. globiferus and V. tuberculatum. In contrary, no protein was detected in all the samples.  Alkaloids  Wagner  +  ---+  +  -----+  -Anthraquinones Borntrager  -------------Cardiac  glycosides   Keller Killani  -------- There was signi cant difference in the percent of total ash values between and within species of mistletoes. The total ash value of E. woodfordiodes and P. regularis ranges from 6.04 to 13.23% and 5.62 to 15.22%, respectively. Comparable total ash content was found in T. globiferus and V. tuberculatum [Table 3]. Similarly, the results on the acid-insoluble ash and water-soluble ash revealed variation between and within species of mistletoes based on the host plants. No signi cant difference was obtained in the mean percent moisture content of the study species.

Discussion
There is an uneven distribution of mistletoes in Africa. Species such as E. woodfordides, P. regularis, and V. tuberculatum are limited to East Africa, and T. globiferus widely distributed from East to West Africa [37].
The results on the phytochemical content of T. globiferus agreed with that reported for the same species by Abubakar et al [14], Bassey [39] and Umarudeen and Chika [40] on anthraquinones, and tannins. In contrast, it partly agrees with results in other phytochemicals to Abubakar et al [14], Bassey [39] and Umarudeen and Chika [40]. On the other hand, the results obtained on alkaloids, quinone and terpenoids in E. woodfordiodes agreed to Ngbolua et al [41]. Similarly, the results obtained in P. regularis partly agrees with P. incana in tannins and anthraquinone [42], tannins, saponin and anthraquinone to P. capitata [43], alkaloid and tannins to P. austroarabica [38]. In general, it is necessary to consider the detection of preliminary phytochemical composition of mistletoe with respect to host plants, extraction solvents [8] and the test methods.
The determination of higher moisture content in this study indicated the more probability of microbial attack and enzymatic hydrolysis of the plant materials [44,[45][46][47]. The higher extraction yield of mistletoes using water and methanol in this study might be associated to the higher polarity of the extraction materials, the particle size of the raw materials, the solvent-to-solid ration, the extraction temperature and the extraction duration [48][49].

Conclusions
This study revealed the presence of secondary metabolites that have potential bioactive activities. Therefore, future studies need to investigate the potential use of these plants for their bioactive compounds using in vitro and in vivo models.

Limitations
This preliminary phytochemical and physicochemical study on mistletoes did not account quanti cation of bioactive compounds and limited to certain part of the country.