Characterization and antimicrobial evaluation of anthraquinones and triterpenes from Rubia cordifolia

Abstract Chemical investigation of roots of the plant, Rubia cordifolia Linn, led to the isolation of an undescribed anthraquinone, cordifoquinone R, determined as 1,2-dihydroxy-6-methoxyanthracene-9,10-dione (6) based on the 1D and 2D NMR analyses and HRESIMS. Ten other known compounds viz.1,4-dihydroxy-2-methoxyanthracene-9,10-dione (1), rubiadin (2), xanthopurpurin (3), 1-methoxy-3-hydroxy-2-carbomethoxy-9,10-anthraquinone (4), alizarin (5), β-sitosterol glucoside (7), scopoletin (8), oleanolic acid, (9), pomolic acid (10), queretaroic acid (11) were also isolated. Out of these compounds, 4, 10, and 11 are first reported from this plant species. Compounds 2, 3, 6, 7, and 10 showed activity in the range of 16-32 µg/ml against S. aureus ATCC 29213.


Introduction
Rubia cordifolia Linn, a well-known ayurvedic herb, belongs to the family Rubiaceae and is commonly known as Indian Madder or manjishtha [1].It is found growing throughout India, mainly in the hilly districts.The plant is used for treating various ailments, viz skin disorders, as a blood purifier, immunomodulant, anti-inflammatoty, etc., in the Ayurvedic system of medicine [2].The folk tribes of West Bengal and Uttaranchal are known to use the roots to treat jaundice [3].It is also an economically important source of natural dyes, especially red pigment.A review of the literature reveals the main chemical constituents of Rubia cordifolia collected in India are anthraquinones, prenyl naphthoquinones, and triterpenes [4].
In continuation of our efforts to identify constituents from plant resources, the phytochemical investigation of R. cordifolia roots was taken up.Column chromatography of the methanolic extract led to the isolation of six anthraquinones, three triterpenes, a coumarin, and a sterol glycoside.Out of these, one anthraquinone, cordifoquinone R (6), is reported for the first time in nature, whereas 1-methoxy-3hydroxy-2-carbomethoxy-9,10-anthraquinone, pomolic acid, and queretaroic acid, to the best of our knowledge, were isolated for the first time from this species.The structures of the compounds were characterized based on spectroscopic studies (IR, NMR, and MS).
Drug resistance amongst pathogenic microorganisms is a growing menace, and identifying new chemical entities capable of inhibiting drug-resistant pathogens is an urgent unmet medical need.Almost 80% of the world population relies on phytopharmaceuticals as a first line of treatment for most microbial infections.Thus, the various phytoconstituents identified here were subjected to antimicrobial and cytotoxicity analysis to determine their applicability as potential new medicines [5].
Compound 6 was isolated as a red-coloured amorphous powder, with m.p. 252-254 C. The molecular formula of C 15 H 10 O 5 was determined based on its NMR and HRESIMS (m/z 271.0611 [M þ H] þ ) data, indicating 11 degrees of unsaturation.The IR spectrum showed signals corresponding to hydroxyl groups at 3447 cm À1 and carbonyl groups at 1671 and 1630 cm À1 .The 1 H and 13 C NMR spectral data of 6 indicated that the compound had an anthraquinone skeleton.The presence of five protons in the aromatic region, along with DEPT 135, which showed the presence of five methine carbons, indicated that the compound was trisubstituted.The 1 H NMR spectrum showed signals corresponding to five aromatic protons, an AMX system at d 8.18 (d, 1H, J ¼ 8.7 Hz, H-8), 7.41 (dd, 1H, J ¼ 8.7, 2.7 Hz, H-7), 7.57 (d, 1H, J ¼ 2.7 Hz, H-5), and two doublets at d 7.20 (d, 1H, J ¼ 8.3 Hz, H-3) and d 7.64 (d, 1H, J ¼ 8.3 Hz, H-4), leading to the conclusion that on one ring there are two substituents ortho to each other, while the third substituent was on a different ring.The signals corresponding to a methoxy group were observed at d 3.96 (s, 3H) and 56.0 in the 1 H and 13 C NMR, respectively, indicating that one of the substituents was a methoxy group.Based on the IR, mass spectra, and appearance of signals corresponding to substituent-bearing quaternary carbons at d C 150.7 and d C 152.9, it could be safely assumed that the remaining two substituents were hydroxyl groups.As the ring C NMR data of 6 was quite similar to that of alizarin [10], an anthraquinone previously reported from R. cordifolia, it could be deduced that the substitution pattern of ring C in compound 6 was the same as that of alizarin, i.e. the two hydroxyl groups were present at C-1 and C-2.However, that still left the assignment of the position of the methoxy group in ring A of compound 6.On comparison of NMR data of 6 with two previously reported anthraquinone, 2,6-dihydroxy-1-methoxy-9,10-anthraquinone (umbellata H) [16] and 1,6-dihydroxy-2-methoxy-9,10-anthraquinone [17], a lot of structural similarities were found.Though these compounds had similar substituent groups as compound 6, the substituent positions were different.Based on the above similarities, we assigned the methoxy group at position 6 in ring A of compound 6.The observance of meta coupling between H-5 and H-7 and ortho coupling between H-8 and H-7 supported this assignment (Figure 1).
All the isolated compounds were tested for antibacterial activity against E. coli ATCC 25922, S. aureus ATCC 29213, K. pneumoniae BAA 1705, A. baumannii BAA 1605, and P. aeruginosa ATCC 27853, with levofloxacin as the standard reference for antibacterial activity (Table 1).As seen in Table 1, compounds 2, 3, 6, 7, and 10 showed activity with 16-32 mg/ml against S. aureus ATCC 29213, respectively, while no compound was active against gram-negative bacterial pathogens tested.
The cytotoxicity of the hits was determined against Vero cells (ATCC CCL-81) to calculate their selectivity indexes (SI), and the data are tabulated in Table 1.As seen above, compounds 2, 3, and 10 were cytotoxic, while compounds 6 and 7 exhibited decent SI to proceed ahead.
To conclude, the chemical investigation of roots of Rubia cordifolia resulted in the isolation of a trisubstituted 9,10 anthraquinone viz.1,2-dihydroxy-6-methoxyanthracene-9,10-dione for the first time in nature and three other compounds viz pomolic acid, queretaroic acid, and 1-methoxy-3-hydroxy-2-carbomethoxy-9,10-anthraquinone for the first time from the plant.Six other previously reported compounds were also isolated from the plant.The antimicrobial and cytotoxicity analysis identified compound 6 exhibiting a decent selectivity index to be pursued further as potential antimicrobial targeting S. aureus.The observance of antimicrobial activity against S. aureus ATCC 29213, a skin disease-causing organism, gives credibility to the ages-old usage of Rubia cordifolia for skin disorders.Given the paucity of novel antimicrobials, this study is a welcome advancement in identifying natural product antimicrobials.

General experimental procedures
Melting points were determined on a Mettler FP 51 melting point apparatus (Mettler Instruments, Switzerland) and are uncorrected.IR spectra were obtained with a Nicolet 740 FT-IR spectrometer (Thermo Electron Corp., Madison, WI, USA).NMR spectra were measured on Bruker AVANCE III HD 400 MHz and Bruker AVANCE III HD 500 MHz spectrometers (Bruker, Fallanden, Switzerland).Chemical shifts (d) are reported in ppm and coupling constants (J) in Hz.Mass spectra were obtained on SHIMADZU LC-MS 8040 (Shimadzu, Manchester, UK) instrument.HRMS were recorded on XEVO G2-XS QTof spectrometer (Waters Corporation, Milford, MA, USA).Column chromatography (CC) was performed on silica gel (60-120 and 100-200 mesh, Merck, Mumbai, India).TLC was performed on pre-coated silica gel 60 F 254 plates (E.Merck, Darmstadt, Germany).NMR grade solvents used were of Sigma Aldrich (St. Louis, MO, USA) make.All the other solvents used were of analytical grade (Merck, Mumbai, India).

Plant material
The roots of Rubia cordifolia were collected from the Tirupati region in the month of April 2012 and identified by Prof. Dr. K. Madhava Chetty, A herbarium voucher (no.0972) has been deposited at the Department of Botany, College of Science, Sri Venkateshwara University, Tirupati, Andhra Pradesh.

Extraction and isolation
Air-dried roots of R cordifolia (4.5 kg) were finely powdered and then subjected to extraction using the percolation method, first with hexane and then followed by methanol, to yield 20 g of reddish hexane extract and 98 g of viscous methanolic extract, after concentration under vacuum.The methanolic extract (80 g) was subjected to column chromatography using silica gel (60-120 mesh) as the stationary phase and eluted initially with 100% hexane followed by gradually increasing the polarity with varying ratio of hexane: ethyl acetate.Different fractions were collected and combined based on their TLC characteristics.Ten fractions were identified, which showed a major component(s), and these were then subjected to re-chromatography on silica gel (100-200 mesh).Fraction 1 (300 mg) was rechromatographed and eluted with hexane-ethyl acetate (5:95), resulting in the isolation of 1 (18 mg).The compounds were screened against a bacterial panel consisting of ESKAPE pathogens, as described previously [5].

Antibiotic susceptibility testing
Antibiotic susceptibility testing was carried out according to the CLSI guidelines for broth micro-dilution assay as described earlier [5].

Cell cytotoxicity assay
Cell toxicity was performed against Vero cells using the MTT assay as described previously [5].

Table 1 .
Antimicrobial activity of the isolated compounds.