# Analysis of total phenolic contents, flavonoids, antioxidant and antibacterial activities of Croton macrostachyus root extracts – BMC Chemistry

May 12, 2022

### Study area description

The study was conducted on selected traditional plants collected from Chiliga, Lay Armachiho, Gondar Zuria and Dembiya Woredas, which ware situated in the Central Gondar Zone of the Amhara regional state (Fig. 1). The elevation of the Woredas ranged from 900 to 2267 m a.s.l. The temperature of the Woredas ranged from 11 to 32 °C with average annual rainfall of 995–1175 mm. The income of the local people is mainly based on sustenance mixed agriculture (crop-livestock production). The study areas were selected based on the large abundance of the plant and dependence of majority of its population on such traditional medicinal plants; and high prevalence of patients infected with rabies who took the root extracts of the plant as traditional medicine could be another reason to mention (Table 1).

### Chemicals and reagents

All the chemicals and reagents used in the present study were of analytical grade. The chemicals and reagents included: Methanol (98%), Folin-Ciocalteu reagent, gallic acid, ascorbic acid, sodium hydroxide, anhydrous sodium carbonate and anhydrous aluminum chloride, which were purchased from Loba Chemie (Mumbai, India); whereas 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium nitrite and catechin were purchased from Sisco Research Laboratories (Mumbai, India).

### Apparatus and equipments

Electrical grinder (IAK–WERKE, Germany), electronic balance (Bosch, Germany), Whatman No. 42 filter paper (110 mm), graduated pipettes, micropipettes (Mumbai, India), refrigerator (Hitachi LR902T, USA), orbital shaker (GEMMY Orbital Shaker, VRN-480, Taiwan), lyophilizer (Scientz-10N, Ningbo Scientz Biotechnology Co., Ltd., China), rotary evaporator (Bibby RE200, Sterilin Ltd., UK) and UV/Vis spectrophotometer (Sanyo SP75, UK) were used in the study.

### Collection of plant material, drying and storage

Identification and authentication of the plant was performed by a Botanist using taxonomic keys and floras, and voucher specimen (002/ATM/2020) was kept at Botanical Science and Herbarium, University of Gondar, for future references. Representative samples of C. macrostachyus root were collected from six sites. Permissions were obtained during plant material collection. From each site, 3 sampling points were selected; two aged plants were selected per sampling point, making a total of six plants per sampling site. Generally, a total of 36 matured plants were selected from 6 sites; and two root samples were collected per plant. Samples were collected from June to September, 2021 (wet season) and January–April, 2021 (dry season) in Ethiopian context. Plant root samples collected from each sampling site were homogenized to get a composite sample after removing surface contaminants by first washing with tap water, followed by distilled water. Here, the root samples were first dried at ambient temperature for three weeks under a plastic cover to avoid dust contamination, powdered using an electrical grinder, sieved through a 100-μm pore size sieve, then homogenized, and kept in clean polyethylene plastic bags until extraction.

### Optimization of extraction conditions

Phenolic contents and antioxidant activity were affected by solvent type, pH level, solvent–water ratio and extraction time [11]. In the present study, solvents ratio (methanol/water) and extraction time were optimized. Accordingly, the solvents ratio was optimized at ambient temperature by changing the methanol/water (v/v) ratio from 50 to 100%, while keeping the extraction time at 24 h. Likewise, the extraction time was optimized by varying the time from 12 to 72 h, while upholding the optimized solvent ratio (80%; v/v) at room temperature. In all cases, 0.5 g of the plant root powder was extracted with 25 mL aqueous methanol.

### Root extracts preparation

The dried and ground powdered root sample (0.5 g) was extracted with 25 mL of 80% aqueous methanol (v/v) by maceration using an orbital shaker at room temperature for 24 h. Then, the extract was filtered through Whatman No. 42 filter paper, and its volume adjusted with 80% methanol. Finally, the filtrate was stored in a refrigerator at 4 °C until further analysis. Extraction was performed in triplicate for each bulk sample and a reagent blank under the same conditions. However, for analysis of antibacterial activities, the methanol extracts were first condensed with a rotary evaporator, whereas the aqueous extracts were lyophilized. The dried extracts were stored with sterilized, sealed-culture tubes at 4 °C until each crude extract was re-constituted with 1 mL of distilled water.

### Preparation of standard solutions

Gallic acid standard was prepared by dissolving gallic acid in 80% methanol; 7.5% Na2CO3, 5% NaNO2, 10% ACl3, 1 M NaOH solutions were prepared with double distilled water. Catechin and ascorbic acid standard solutions were also prepared with methanol.

### Total polyphenolic content

Total phenolic concentration in the root extract was spectrophotometrically determined by the Folin–Ciocalteu assay, using gallic acid as a standard [17, 18]. The reaction mixture was prepared by mixing 0.5 mL of plant root extract, 3 mL of distilled water and 0.25 mL of Folin-Ciocalteu reagent and shaken. After 5 min in the dark, 1 mL of 7.5% Na2CO3 was added and incubated at room temperature for 90 min in the dark. Reagent blank was also parallelly prepared using distilled water. The absorbance was measured against the prepared reagent blank at 760 nm using a double beam UV/Vis spectrophotometer. The concentration of total phenolic compounds in the extract was expressed as milligram of gallic acid equivalent (GAE) per 100 g sample (mg GAE/100 g). All the samples were analyzed in triplicate.

### Determination of flavonoid content

Flavonoid content was determined using aluminum chloride assay according to [18]. Briefly, an aliquot (0.5 mL) of the extract was added to a 10 mL test tube containing 2 mL of distilled water. To each test tube, 0.15 mL of 5% NaNO2 was added. After 5 min of incubation, 0.15 mL 10% AlCl3 was added. After 1 min, 1 mL of 1 M NaOH was added and the volume was adjusted to 5 mL with distilled water. After 10 min, the absorbance of the resulting solution was measured at 510 nm. Catechin was used as standard to express total flavonoids contents of samples as mg catechin equivalent per 100 g of sample (mg CE/100 g sample). All the samples were analyzed in triplicate.

### Determination of antioxidant activity

The antioxidant activity of the plant root extracts was evaluated using the DPPH method as described by [19] with slight modification. A mass of 20 mg of DPPH was dissolved with a small amount of methanol in a 500 mL volumetric flask. After the DPPH was fully dissolved, the flask was filled up to the mark with methanol. The control was prepared by mixing 3 mL of methanol and 2 mL of DPPH solution. In addition, a stock solution of ascorbic acid was prepared by dissolving 50 mg of ascorbic acid in 100 mL of methanol. Generation of calibration curve was achieved by preparing different concentrations, viz. 1, 2.5, 5, 10, 15, 20, 25, 50, 100, 150 and 200 mg/L, from the stock solution. To each test tube, 3 mL of standard ascorbic acid and 2 mL of DPPH solutions were added; then the test tubes were covered by aluminum foil and kept in the dark for 30 min. For the samples, a 0.1, 0.2, 0.4, 0.8, 1.6 and 2.4-mL portions of the extracts were mixed with 1.6 mL of DPPH and the final volume of each solution was adjusted to 4 mL with 80% aqueous methanol. The mixture was kept in the dark for 30 min. Finally, the absorbance was measured at 517 nm. The percentage of DPPH radical scavenging activity was determined using the formula:

$$% {text{ Inhibition}} = left[ {{{left( {{text{A}}_{{{text{DPPH}}}} – {text{A}}_{{{text{Sample}}}} } right)} mathord{left/ {vphantom {{left( {{text{A}}_{{{text{DPPH}}}} – {text{A}}_{{{text{Sample}}}} } right)} {{text{A}}_{{{text{DPPH}}}} }}} right. kern-nulldelimiterspace} {{text{A}}_{{{text{DPPH}}}} }}} right] times 100.$$

where ADPPH was absorbance of DPPH control solution and ASample was absorbance of DPPH solution in the presence of plant extract. Sample concentration giving 50% inhibition was estimated as IC50 value using the dose inhibition curve in linear range by plotting the extract concentration versus the corresponding scavenging activity. Measurements were performed in triplicates. The results were expressed as mg ascorbic acid equivalent per gram of sample (mg AAE/g sample).

### Evaluation of the antibacterial activity

The disk diffusion method was used to evaluate antimicrobial activity of the plant root extracts against four bacterial strains, namely: gram-positive bacteria species (Staphylococcus aurous and Staphylococcus pneumonia) and gram-negative bacteria species (Escherichia coli and Klebsiella pneumonia) using Gentamicin disc as standard drug [6]. These microorganisms were cultured at the molecular biology laboratory, Department of Biology, University of Gondar.

The media were prepared according to the manufacturer’s instruction as follows: 38 g of the Muller Hinton agar powder was dissolved in 1000 mL of distilled water, then heated, shaken well and allowed to boil and completely dissolved. Then, it was placed in an autoclave at 15 Pa pressure (121 °C) for about 15 min to sterilize the media. After that, the media were cooled, poured into 12 plates and put on a leveled surface. Lastly, the media were allowed to solidify, and kept in a laminar air flow hood in an upright position to avoid contamination. Lastly, the test culture bacteria were swabbed on the top of the pre-leveled media and allowed to dry. A sterilized Pasteur pipette was used to bore holes on plates and 100 μL of each extract was applied to the holes in triplicate using Gentamicin drug as positive control. The petri dish was incubated at 37 °C for 24 h. At the end of incubation period, the antibacterial activities of root extracts were determined by measuring the average inhibition zones of the extract and positive control in radius millimeter.

### Statistical analysis

All statistical analyses were undertaken using SPSS Version 20. Differences at p < 0.05 were significant.