Anthraquinones from the Roots of Kniphofia insignis and Evaluation of Their Antimicrobial Activities

Sequential extraction using a cold maceration method and column chromatographic separation of the roots Kniphofia insignis headed to the isolation of three anthraquinones: one monomeric anthraquinone (1) and two dimeric anthraquinones (2 and 3). It was further purified by Sephadex LH-20 and recrystallized. 'e structures of these compounds were established based on the spectroscopic analyses including NMR (H-NMR and C-NMR and infrared) and comparison with reported literatures. In an in vitro antimicrobial assay of the crude extracts, the isolated compounds were made against four bacterial strains (S. aureus ATCC 25923, B. subtilis ATCC 6633, E. coli ATCC 35218, and P. aeruginosa ATCC 27853) and Fusarium spp. fungal strain. In the crude extracts of chloroform, substantial antimicrobial activity was seen with the highest activity against B. subtilis (16mm) and E. coli (22mm). Meanwhile, compound 1 has a better zone of inhibition with 14mm against P. aeruginosa,whereas compound 2 showed better activity (13mm) against Fusarium spp. fungal strain.


Introduction
e genus Kniphofia Moench (family Asphodelaceae) is commonly named as "red hot pokers," containing about 71 species. About seven species of Kniphofia occur in Ethiopia, of which five including K. foliosa, K. hildebrandtii, K. isoetifolia, K. insignis, and K. schimperi are endemic [1,2], and the genus is widely known for its ornamental value due to their colorful flowers and used in traditional medicine [3]. Its roots are used to treat abdominal cramps, wound healing, women infertility, gonorrhea, hepatitis B, ring worm, chest pain, snake bite, and shoulder pains [1,[3][4][5][6]. e family Asphodelaceae is a rich source of mainly anthraquinones, monomeric and dimeric anthraquinones, anthrones, dimeric phenylanthraquinones, [5] and naphthoquinone [4]. It is so far only known from the Shewa and Arsi Zone. e main flowering period is from June to September, and it is commonly seen in the Sululta plains between Addis Ababa and Chancho [2]. However, it is a phytochemical investigation, and the antimicrobial activity evaluation has not been reported yet. erefore, herein, we purpose the current project to carry out isolation and characterization of compounds from the root of Kniphofia insignis and evaluate their antimicrobial activities.

General Information.
Analytical grade solvents such as n-hexane, chloroform, acetone, methanol, and ethyl acetate were used for extraction and column elution; silica gel 60-120 mm mesh size, oxalic acid, TLC silica gel coated plate for detection of spots, CDCl 3 for recording NMR spectra, and DMSO for sample preparation for antibacterial susceptibility test, standard antibiotic drug (gentamycin and chlotrimazole), Mueller-Hinton agar, nutrient agar, and saline solution were used as a culture medium during the antibacterial and antifungal test, and Sephadex LH-20 was also used for further purification. Round-bottom flask of sizes 50, 100, and 500 mL, measuring cylinder, Whatman No. 1 filter paper, pistil and mortar, weighing balance, column chromatography, and rotary evaporator were used during extraction and purification. UV chambers of 254 and 365 nm (LF-260.LS, EEC) were used for detection of spot. An infrared (IR) spectrum was measured using the Perkin-Elmer IR spectrophotometer. e 1D ( 1 H-NMR (400 and 400 MHz) and 13 C-NMR (125 MHz)) spectra were recorded using Bruker Avance NMR in deuterated solvent were used for characterization.

Collection and Preparation of Plant Material.
e roots of Kniphofia insignis were collected from Ethiopia, North Shewa Zone Oromia Regional State, Jida District, which is about 116 km away from Addis Ababa. It was collected, airdried, and powdered so as to allow the penetration of the solvents during the extraction and stored in an appropriate container in Jimma University Organic research laboratory. e plant material was identified by Jimma University Botanist Dr. Dereje Denu, and the voucher specimen (voucher number CH1) has been deposited in Jimma University Herbarium.
Based on its bacterial activities test and TLC profile, 23 g of chloroform extract was adsorbed on silica gel (60-120 mm mesh size) and subjected to 500 mm diameter column chromatography on silica gel (165 g) eluting with n-hexane with increasing amounts of ethyl acetate gradient. e following ratios of solvent combinations were used in the elution process in n-hexane to ethyl acetate ratio of 100 : 0 to 0 : 100. e main fractions which yield one pure compound at 1% of ethyl acetate in n-hexane were followed by further purification using Sephadex LH-20 in provided compound 1 (32 mg). e other fraction eluted with 5% of ethyl acetate in n-hexane yields two dimeric anthraquinones: asphodeline (2, 4.3 mg) and 10-hydroxy-10-(chrysophanol-7′-yl)-chrysophanol anthrone (3, 3 mg) up on further purification using small column chromatography. e isolated compounds were characterized by the spectroscopic techniques: FT-IR, 1 H-NMR and 13 C-NMR for compound 1, and 1 H-NMR and using reported 13 C-NMR for compounds 2 and 3 at Addis Ababa University.

Antimicrobial Assay.
e crude extracts and isolated compounds were evaluated for in vitro antimicrobial activities against four bacteria strains (E. coli ATCC 35218, S. aureus ATCC 25923, B. subtilis ATCC 6633, and P. aeruginosa ATCC 27853) and one fungus strain (Fusarium spp.) by the agar disc diffusion method. e antimicrobial activity test was done using the agar disc diffusion method following the standard procedures [7][8][9]. e stock solution was prepared by dissolving 100 mg of the crude extract in 1 mL DMSO, 25 mg of the compound 1 in 0.5 mL, 4 mg of compound 2 in 0.2 mL to get 100 mgmL −1 of crude extracts, and 50 and 20 mgmL −1 final stock solutions of 1 and 2 pure compounds, respectively. Also, the inhibition zone was measured in mm after 24 h of incubation for bacteria and 48 h for fungus at 37°C, and compared with the standard drug, gentamycin for bacteria and clotrimazole for fungus, the inhibition diameter was measured.
Compound 2 (4.3 mg) was isolated as a red crystal from 5% ethyl acetate in n-hexane. e 1 H-NMR (Table 1) spectrum showed the presence of two aromatic methyl groups resonating at δ H 2.33(3H, s, 3-CH 3 ) and 2.53(3H, s, 3′-CH 3 ), eight aromatic protons, and four chelated hydroxyl groups at δ H (12.00, 12.09, 12.45, and 12.53), which confirmed that this compound is dimeric anthraquinone. In one half of the molecule, the 1 H-NMR spectrum showed three mutually coupled aromatic protons in the ABX spin system   Table 1).
e crude extracts showed considerable activity on both Gram-positive and Gram-negative bacterial strains with zone of inhibition ranging from 12 to 22 mm, with the highest activity (22 mm) observed for chloroform extract against E. coli. However, the inhibitions displayed on both Gram-negative and Gram-positive bacteria for the isolated compounds that have been tested were good with variable degree of potency. e better activity of the crude extracts over the isolated compound could be accounted to the synergistic interactions of several compounds present in the extract, which cannot be the case when single compounds are evaluated. Acetone extract showed the highest inhibition (18 mm) against the fungal strain, Fusarium spp., and lowest by hexane extract (8 mm), whereas compound 2 showed a higher inhibition (13 mm) as compared to compound 1 against the same strain.

Data Availability
All data used to support the findings of this study are available in the manuscript.