Plant Material Collection and Processing
Full-grown wild plants were selected from Dara District (6° 41′ 94.39′′ N, 38° 31′ 8.198′′ E), Sidama Region, Ethiopia. Tariku Berihun (PhD) a botanist at Dilla University confirmed taxonomical identification of the plant using Flora of Ethiopia and Eritrea Vol. 03, Page 102-105 [26]. The pressed plant spacemen were stored in Dilla University's publicly available herbarium. Test plants were collected and dried in the shade and at ambient temperature on a clean paper magazine for two weeks [27,28]. Subsequently, they were ground using a coffee bean grinding machine and sifted through a 200µm mesh. The powdered samples were stored in a tightly closed plastic envelope. The collection of the plant material and related research complies with relevant institutional, national, and international guidelines and legislation.
Plant Extraction
The maceration technique was utilized in the extraction process. For aqueous extraction, 1 g of plant leaves and pod powder was saturated in 1000 ml of cold distilled water in the flask, shaken for 24 hours on an orbital shaker at 110 rpm, and then directly used as a stock solution of 1000 ppm [29].
For ethanol extraction, 150 g of leaves and 100 g of pod powder were soaked in 1.5 and 1 liters of 97% ethanol, respectively, in a 1:10 ratio [30], in an Erlenmeyer flask of 500 ml volume. The solutions were shaken for 24 hours in an orbital shaker at 125 rpm. The solutions were filtered using Whatman filter paper. The filtrates were then evaporated in a rotary evaporator at a temperature below 40 °C [31]. Finally, the extracts were labeled and stored until needed.
Preliminary phytochemical screening
The preliminary qualitative phytochemical identification of the crude ethanol extract of C. aurea leaves and pods were carried out using standard tests performed according to Kenubih et al. [32] and Mulata et al. [33].
Alkaloids
To identify alkaloids, the Mayer's test was performed. Briefly, 0.2 g of extracts were added to each test tube, followed by 3 ml of hexane, vigorously agitated, and filtered. A test tube was filled with 5 milliliters of 2% hydrochloric acid (HCL). After boiling and filtering, a few drops of picric acid were added to the liquid. The production of a yellow precipitate suggests the presence of alkaloids.
Anthocyanin
A 1 g sample of each solvent extract was mixed with 5 ml of HCL and filtered. A 5ml solution of 10% ammonium hydroxide was added to the filtrate and thoroughly shaken. Pink, red, or violet colors in the ammoniac phase were regarded as a sign of anthocyanin.
Flavonoids
1 ml of plant extract was mixed with a few drops of 10% lead acetate solution. A yellow precipitate indicated the presence of flavonoids.
Phenolic compounds
In a test tube, 200 mg of phthalic anhydride was added to the extract, followed by a few drops of strong sulfuric acid. The solution was gently heated for 2-3 minutes. After cooling, the mixture was poured into a beaker containing diluted sodium hydroxide solution and diluted with an equal amount of water. A yellowish precipitate indicated the presence of phenolic compounds.
Tannins
In a test tube, 0.25 g of each solvent extract was heated in 10 ml of distilled water. After boiling, the mixture was filtered and a few drops of 0.1% ferric chloride were added to the filtrate. The formation of a blue-black or greenish-black precipitate indicated the presence of tannins.
Terpenoids
Two milliliters of chloroform were combined with 0.25 gram of each extract. Then, 3 mL of pure sulfuric acid was carefully applied to create a coating. The reddish-brown coloring of the interface showed the presence of terpenoids.
Saponins
To test for saponins, 0.5 g of each extract was boiled with 5 ml of distilled water and then filtered. The filtrate was shaken vigorously. The formation of stable foam indicated the presence of saponins
Steroids
2 g of extract is diluted in 2 mL of acetic anhydride and 1-2 drops of strong sulfuric acid (H2SO4). The combination begins as pink, but as the reaction develops, it turns blue. Finally, it could seem green. This signaled the existence of steroids.
Tick Collection, and acclimatization
Tick A. variegatum and R. microplus were collected from cattle that were brought to a veterinary clinic located at (6°47′75.91″ N, 38°34′2.261″ E) and from naturally infested cattle pastured in a local grazing area (6°47′73.83″ N, 38°28′4.311″ E) Dara District, Sidama Region, Ethiopia. The samples were then placed in a plastic box lined with cotton wool and sealed with nylon mesh [34,35]. When submitting acaricides, it was checked to ensure that none had been used in the previous 45 days. The insects were then carried to the Dilla University insectary with care, keeping them away from the hot engine of the car to prevent die-off. Ticks were identified and recorded using a stereomicroscope within a few hours of arrival [10,35].
Adult ticks were acclimatized by being kept in vials with open tops and fully covered with a piece of nylon mesh to ensure protection, sufficient airflow, and humidity. Males and females were stored separately to prevent inbreeding. All vials containing ticks were kept in a plastic box inside environmental chambers (incubators) at 22 °C ± 1°C and 12 hr:12 hr day and night for one week [36].
Rearing
Engorged female ticks were washed with distilled water and dried upon arrival. The plastic box that is full of watered-down sand was prepared. Up to five clean, engorged female ticks were placed in a beaker, and the beaker was buried in the sand until the sand-covered half of the beaker was in the plastic box. Incubated at 27 ± 1°C and 85 ± 10 % relative humidity. Under optimal rearing conditions, the engorged female ticks of most species begin to lay eggs within 2–7 days. All eggs were collected in a vial seven days after the commencement of incubation. Each vial containing the first week’s egg production was labeled with the date, to make the selection more uniform [37].
Test Bioassay Preparation
1 gram of dry extract and 1000 ml of dechlorinated water were mixed to prepare a 1000 ppm stock. Then, 80 ml of serial dilutions of 12.5, 25, 50, 100, 200, and 400 ppm were prepared in clean beakers. Distilled water was used for negative control and 0.1% diazinon® (Adamitulu Pesticide Processing S.Co., Zeway, Ethiopia) for positive control [37].
Adult immersion test
The adult immersion tests (AIT) were implemented according to Kenubih and Fouche [32,37] with some modifications for acaricidal activity tests of crude extracts of plant materials. Ten adult ticks were exposed to each dilution in a clean Petri dish for 10 minutes by immersion. Positive and negative controls were prepared in the same manner. The test was set up in three replicates [38,39]. Then they were picked out, washed in tap water, and subsequently transferred to another sterile Petri dish and incubated for 24 hours. with an average humidity of 80 ± 10% at 22°C ± 1°C [40]. Finally, dead and live ticks were counted through careful observation under a stereomicroscope. The ticks were judged dead if there were no signs of movement at all or signs of cuticle darkness [37].
Egg immersion test
Envelopes of filter paper (Whatman No. 1) were prepared, and twenty 15-day-old reared eggs were placed in each envelope. The samples were then immersed in prepared serial dilutions for 10 minutes in distilled water, and diazinon was used for the respective negative and positive controls. Finally, the solution was decanted and evaporated from the envelope. Treated eggs were placed in vials and incubated at 28 ± 1 OC and 85 ± 5% for 15 days until larvae hatching was completed [38,39]. Each treatment in the experiments was repeated three times. Finally, hatched larvae and unhatched eggs were identified and counted using a stereomicroscope with a range of magnification from x10 to x40.
Data Analysis
Mortality and hatchability data were analyzed using SPSS software version 20. Mean ± standard error (Mean ± SE) expressed by one-way analysis of variance (ANOVA) with multiple comparison tests (Tukey’s test) to determine a significant mean mortality and hatchability difference of the concentration, while estimation of the median lethal concentration, LC50, and inhibition concentration, IC50, was made by the probit regression model. The toxicity level of plant extract was classified as follows: non-toxic (IC and LC50 > 1000 ppm); less toxic (IC and LC50 = 500–1000 ppm); moderately toxic (IC and LC50 = 100–500 ppm); strongly toxic (IC and LC50 < 100 ppm), according to Fouche et al. [37].