Journal of Chemistry
Volume 2021 (2021), Article ID 6661116, 5 pages
https://doi.org/10.1155/2021/6661116

Research Article

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

Asnakew Amare Tadesse,¹ Negera Abdissa Ayana,² and Dele Abdissa Keneni²

¹Department of Chemistry, College of Natural Sciences, Woldia University, P.O. Box 400, Woldia, Ethiopia
²Department of Chemistry, College of Natural Sciences, Jimma University, P.O. Box 378, Jimma, Ethiopia

Correspondence should be addressed to Asnakew Amare Tadesse; asnamare6@gmail.com

Received 26 December 2020; Revised 26 March 2021; Accepted 2 April 2021; Published 14 April 2021

Academic Editor: Liviu Mitu

Copyright © 2021 Asnakew Amare Tadesse et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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. The structures of these compounds were established based on the spectroscopic analyses including NMR (¹H-NMR and ¹³C-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 (16 mm) and E. coli (22 mm). Meanwhile, compound 1 has a better zone of inhibition with 14 mm against P. aeruginosa, whereas compound 2 showed better activity (13 mm) against Fusarium spp. fungal strain.

1. Introduction

The 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, 3–6]. The 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. The 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. Therefore, 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.

2. Experimental Section

2.1. 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, CDCl₃ 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. The 1D (¹H-NMR (400 and 400 MHz) and ¹³C-NMR (125 MHz)) spectra were recorded using Bruker Avance NMR in deuterated solvent were used for characterization.

2.2. Collection and Preparation of Plant Material

The 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, air-dried, 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. The 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.

2.3. Extraction and Isolation

The shaded dried roots (1 kg) of powdered K. insignis were subjected to sequential extraction with n-hexane, chloroform, acetone, and methanol using the cold maceration technique three times for 24 h each with 2.5 L at room temperature. Then, the crude extracts were concentrated using a rotary evaporator so as to remove the solvent to yield 10 g (1%), 27.5 g (27.5%), 17 g (1.7%), and 45 g (4.5%) n-hexane, chloroform, acetone, and methanol extracts, respectively. The crude extracts were subjected to antimicrobial activities against four bacterial strains (S. aureus (ATCC 25923), E. coli (ATCC 35218), P. aeruginosa (ATCC 27853), and B. subtilis (ATCC 6633)) and fungal strain (Fusarium spp.).

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. The following ratios of solvent combinations were used in the elution process in n-hexane to ethyl acetate ratio of 100 : 0 to 0 : 100. The 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). The 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. The isolated compounds were characterized by the spectroscopic techniques: FT-IR, ¹H-NMR and ¹³C-NMR for compound 1, and ¹H-NMR and using reported ¹³C-NMR for compounds 2 and 3 at Addis Ababa University.

2.4. Antimicrobial Assay

The 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. The antimicrobial activity test was done using the agar disc diffusion method following the standard procedures [7–9]. The 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.

3. Results and Discussion

3.1. Characterization of Isolated Compounds

Compound 1 (32 mg) was isolated as red amorphous solid. It was isolated with 1% of ethyl acetate in n-hexane, and its Rf value was 0.87 in 95% n-hexane in ethyl acetate. Its FT-IR spectral analysis showed a strong band in the region of 3426 and 2917 cm⁻¹ for the hydroxyl (OH) stretching and aromatic (C–H) stretching vibrations, respectively. Other characteristic signals for carbonyl (C=O) bond vibrations and strong C–O bond stretching were also observed at 1616 and 1268 cm⁻¹, respectively.

¹H-NMR spectrum (400 MHz, CDCl₃) (Table 1) displayed three –OH protons at δH 13.04, 12.36, and 12.17 involved in hydrogen bonding and was assigned to –OH groups at C-5, C-8, and C-1, respectively, of an anthraquinone skeleton. In ring A, two meta-coupled aromatic protons at δH 7.14 (1H, d, J = 1.8, H-2) and 7.73 (1H, d, J = 4 Hz, H-4) were observed in addition to the biosynthetically expected methyl group (δH 2.51; δC 22.3 at C-3; δC 149.1), whereas, in ring C, there were two ortho-coupled equivalent aromatic protons resonating at 7.32 (2H, d, J = 8.0 Hz) which were assigned to H-6 and H-7, confirming the presence of chelated –OH group at δH 13.04 being at C-5 (δC 158.3).

Table 1: ¹H-NMR (400 MHz, CDCl₃) and ¹³C-NMR (125 MHz) spectral data of compounds 1, 2, and 3.

Position
1
2
3

δH (m, J in Hz)
δC
δH (m, J in Hz)
δC [10]
δH (m, J in Hz)
δC [11]

1
—
162.9
—
162.9
—
161.8

1′
—
—
—
162.2
—
161.9

1a
—
114.0
—
114.8
—
112.6

1′a
—
—
—
113.9
—
114

2
7.14 (1H, s)
124.6
7.73 (1H, s)
125.6
6.83 (1H, d, J = 4)
117.2

2′
—
—
7.16 (1H, s)
121.4
7.06 (1H, d, J = 4)
124.3

3
—
149.1
—
149.0
—
148.9

3′
—
—
—
149.2
—
149.6

3′-CH₃
—
—
2.53 (3H, s)
149.2
2.47 (3H, s)
22.0

4
7.73 (1H, d, 4)
120.8
—
137.3
6.63 (1H, brs)
120.9

4a
—
133.2
—
132.8
—
148.4

4′a
—
—
—
134.0
—
133.3

4′
—
—
7.87 (1H, s)
120.4
7.66 (1H, d, J = 4)
120.9

5
—
158.3
7.88 (1H, d, J = 8)
124.3
6.80 (1H, d, J = 4)
119.9

5a
—
112.5
—
130.4
—
148.3

5′a
—
—
—
131.1
—
132.8

5′
—
—
8.0 (1H, d, J = 8)
120.0
8.03 (1H, d,J = 8)
119.3

6
7.32 (1H, d, 8.0)
129.5
7.73 (1H, s)
137.2
7.43 (1H, t, J = 8)
137.3

6′
—
—
7.65 (1H, d, J = 8)
135.0
8.67 (1H, d, J = 8)
132.2

7
7.32 (1H, d, 8.0)
129.6
7.34 (1H, d, J = 8)
124.2
6.98 (1H, d, J = 8)
116.9

7′
—
—
—
133.5
—
142.2

8
—
157.6
—
162.8
—
161.5

8′
—
—
—
159.7
—
159.1

8a
—
112.8
—
115.7
—
114.7

8′a
—
—
—
115.5
—
115.9

9
—
190.6
—
192.8
—
193.0

9′
—
—
—
192.8
—
192.1

10
—
186.6
—
182.4
—
70

10′
—
—
—
181.9
—
181.5

3-CH₃
2.51 (3H, s)
22.3
2.33 (3H, s)
22.3
—
22.2

1-OH
12.17 (1H, s)
—
12.53 (1H, s)
—
11.81 (1H, s)
—

1′-OH
—
—
12.45 (1H, s)
—
12.38 (1H, s)
—

5-OH
13.04 (1H, s)
—
—
—
—
—

8-OH
12.36 (1H, s)
—
12.09 (1H, s)
—
12.18 (1H, s)
—

8′-OH
—
—
12.00 (1H, s)
—
12.48 (1H, s)
—

The ¹³C-NMR spectral data (Table 1) revealed the presence of fifteen carbon signals including two carbonyl carbons (δC 190.6 and 186.6), four aromatic methine carbons (δC 129.6, 129.5, 124.6, and 120.8), three oxygenated aromatic quaternary carbons (δC 162.9, 158.3, and 157.6), five aromatic quaternary carbons (δC 149.1, 133.2, 114, 112.8, and 112.5), and a methyl carbon (δC 22.3). Therefore, compound 1 was identified as 1,5,8-trihydroxy-3-methylanthraquinone, trivial name helminthosporin (1) (Figure 1), which has been previously reported from Aloe dawei and Aloe lateritia subspecies graminicola [12], but it was reported for the first time from the genus Kniphofia.

Figure 1: The structures of helminthosporin (1), asphodeline (2), and 10-hydroxy-10-(chrysophanol-7′-yl)-chrysophanol anthrone (3) isolated from root of K. insignis.

Compound 2 (4.3 mg) was isolated as a red crystal from 5% ethyl acetate in n-hexane. The ¹H-NMR (Table 1) spectrum showed the presence of two aromatic methyl groups resonating at δH 2.33(3H, s, 3-CH₃) and 2.53(3H, s, 3′-CH₃), eight aromatic protons, and four chelated hydroxyl groups at δH (12.00, 12.09, 12.45, and 12.53), which confirmed that this compound is dimeric anthraquinone. In one half of the molecule, the ¹H-NMR spectrum showed three mutually coupled aromatic protons in the ABX spin system at δH 7.88 (1H, d, J = 8 Hz), 7.73 (1H, s), and 7.34 (1H, d, J = 8 Hz) assigned for H-5, H-6, and H-7, respectively, of the ring C and singlet proton resonated at δH 7.73 (1H, s, H-2) (Table 1).

Similarly, in the other half of the molecule (ring A'B'C′), the ¹H-NMR spectrum displayed that the broad singlet proton at δH 7.16 (1H, s) and δH 7.87 (1H, s) in ring C′ was assigned to H-2′ and H-4′, respectively, with biosynthetically expected methyl group being at C-3′. In ring A′, two ortho-coupled aromatic protons with AX spin system were resonating at δH 8.0 (1H, d J = 8 Hz) and 7.65 (1H, d, J = 8) for H-5′ and H-6′, respectively, leaving C-7′ for point of attachment to the first chrysophanol moiety (ring ABC). Thus, the structure of compound (2) was found to be 4,7′-bichrysophanol, trivial name asphodeline (2). As reported so far, it is the common metabolite from the genus Kniphofia including K. albescens, K. linearifolia, K. ensifolia, K. evansii, K. northiae, K. foliosa, K. tysonii, and K. isoetifolia [4, 13].

Compound 3 (3 mg) was isolated as red crystal. The ¹H-NMR data of 3 (Table 1) confirmed that this compound is also dimeric anthraquinone. However, its spectral data were similar to compound 2 with one additional aromatic proton due to the difference position of dimerization. Two aromatic methyl signals at δH 2.28 (3H, s, 3-CH₃) and 2.47(3H, s, 3′-CH₃) were observed for the two aromatic methyl groups and four chelated –OH groups at δH (12.48, 12.38, 12.18, and 11.81). Three mutually coupled aromatic protons in the ABX spin system at δH 6.80 (1H, d, J = 4 Hz, H-5), δH 7.43 (1H, t, J = 8 Hz, H-6), and δH 6.98 (1H, d, J = 8 Hz, H-7) were observed. Two meta-coupled aromatic protons at δH 6.83(1H, d, J = 4 Hz, H-2) and 6.63 (1H, brs, H-4) were observed. The remaining spectral data for the other half of the molecule ring A′B′C′ system of 3 are similar to that of 2; two meta-coupled proton signals at δH 7.06 (1H, d, J = 4 Hz, H-2′) and δH 7.66 (1H, d, J = 4 Hz, H-4′) and two ortho-coupled aromatic protons with AX spin system at δH 8.03 (1H, d, J = 8 Hz, H-5′) and δH 8.67 (1H, d, J = 10 Hz, H-6′) were observed.

The first chrysophanol moiety showed an ABX spin system for H-5, H-6, and H-7; the second chrysophanol moiety showed an AX spin system for H-5′ and H-6′ and leaves H-7′ for point of attachment to the first chrysophanol moiety. Deshielded H-4′ (δH 7.66) and H-5′ (δH 8.03) (ring A’B’C′) in contrast to H-4 (δH 6.63) and H-5 (δH 6.80) (ring ABC) indicates the second moiety has a carbonyl group located at C-10’. Therefore, compound 3 was characterized and identified as 10-hydroxy-10-(chrysophanol-7′-yl)-chrysophanol anthrone, which was previously reported from other Kniphofia species such as K. isoetifolia [4], K. ensifolia [14, 15], K. foliosa, and K. thomsonis [10].

3.2. Evaluation of Antimicrobial Activity

The antimicrobial activity of the extracts (100 mg·mL⁻¹) and the isolated compounds 1 (50 mg·mL⁻¹) and 2 (25 mg·mL⁻¹) is shown in Table 2.

Table 2: Zone of inhibition of the extracts and compound 1 and compound 2 in mm.

Test organism
Crude
Extract
Pure

Hexane
Chl
Ac
Meth
1
2
G
C
DMSO

E. coli
16
22
18
16
14
13
32
—
NI

P. aeruginosa
12
15
14
12
15
12
22
—
NI

S. aureus
13
13
15
14
12
14
33
—
NI

B. subtilis
13
16
18
15
13
11
31
—
NI

Fusarium spp.
8
12
18
14
12
13
—
20

Key. Chl = chloroform; Ac = acetone; Meth = methanol; G = gentamycin; NI = no inhibition; C = chlotrimazole CH₃Cl.

The 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. The 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.

4. Conclusion

Phytochemical investigation of the roots of Kniphofia insignis has resulted in identification of three anthraquinones, namely, helminthosporin (1), asphodeline (2), and 10-hydroxy-10-(chrysophanol-7′-yl)-chrysophanol anthrone (3). From its sequential extraction, 10, 28, 17, and 45 g of n-hexane, chloroform, acetone, and methanol crude extracts were obtained, respectively. Its antimicrobial evaluation has revealed that the extracts have almost a better activity against the strains. The antimicrobial analysis showed that the chloroform extract showed the highest inhibition against E. coli.

Data Availability

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

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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