Luteolin and Kaempferol From Cassia Alata

LUTEOLIN AND KAEMPFEROL FROM CASSIA ALATA, ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF ITS METHANOLIC EXTRACTS
ANEELA WAHAB, TAHIRA, SABIRA BEGUM, ANJUM AYUB, IFFAT MAHMOOD, TALAT MAHMOOD, AQEEL AHMAD AND NIDA FAYYAZ
 
Abstract
Cassia alata also known as candlebush is a medicinally important plant. In the present investigation we are reporting the isolation and structure elucidation of two flavonoids kaempferol (1) and luteolin (2) isolated from methanolic extract of its beans through bioassay guided fractionation. The structure of isolated compounds were characterized by spectroscopic techniques such as EIMS, 1H-NMR and 13C-NMR. In this article we are also presenting the antibacterial, antifungal and antioxidant activity of the methanolic extract of its leaves (CA-L), stem (CA-S) and beans (CA-BN). All the extracts showed remarkable antibacterial and weak antioxidant activity whereas moderate antifungal activity was only found in stem (CA-S) and beans (CA-BN) extracts.
Introduction
Cassia alata (Synonym; Senna alata) belonging to the family Leguminosae and subfamily of Fabaceae, commonly known as seven golden candlesticks, and ringworm senna (Quattrocchi U., F.L.S., 2012). This plant is native to the West Indies, tropical America, found wild almost throughout India and Pakistan (Khare C.P., 2007). C. alata with golden blooms is a summer bloomer and a striking spring that last for several weeks but prefer cooler month for flowering (Ray A.B., et al., 2010, Krishnan M. K. S., 1992). This shrub may grow up to 3 meters tall with irregular, angled, glabrous branches. Flowers have bright yellow colour. It has long, membranous, dehiscent pods with 25 or more seeds per pod (Ross I.A., 2003, Bhattacharjee S.K., 2004).

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Cassia alata is widely used as traditional medicine in India and Southeast Asia ( Reezal I., et al., 2002 ). This plant is reported to possess insecticidal, anti-inflammatory, hydragogue, sudorific, diuretic, pesticidal properties. Fresh leaves juice is used for ring worm, snakebite, scorpion bite, skin diseases, impetigo, syphilis sores, itching, mycosis (washerman’s itch), herpes and eczema. Roots, leaves and flowers of this plant possess many biological properties such as antibacterial, antifungal, anti-inflammatory, antitumor, expectorant and also useful in urinary tract problems (Quattrocchi U., F.L.S., 2012), asthma, bronchitis and constipation (Joshi S.G., 2000). The ethyl acetate extract of C. alata leaves possess hypoglycaemic activity (Ray A.B., et al, 2010). This plant also has hepatoprotective property. The main constituents of C.alata are flavonoids, alkaloids, anthraquinone derivatives, tannins, sterols and triterpenes (Neharkar V.S., Gaikward K.G., 2011). The present paper describe the isolation and characterization of kaempferol (1) and luteolin (2). Herein we are also reporting the antimicrobial and antioxidant activities of the methanolic extract of leaves, stem and beans of this plant. All the extracts showed significant antibacterial (Table 2) and weak antioxidant activity (Table 4). Antifungal activity (Table 3) was only observed in the extract of stem and beans.
Experimental
Materials and Methods
General:
Silica gel PF254 (Merk) was used for vacuum liquid chromatography (VLC). Thin layer chromatography (TLC) was performed on pre-coated silica gel F254 (Merck). Gel permeation chromatography was performed on sephadex LH-20 (Pharmcia). The EIMS (electron impact mass spectrometery) were scanned on Jeol-JMS HX-110 mass spectrormeter. The 1H and 13C-NMR (Nuclear Magnetic Resonance) spectra were recorded on a Bruker spectrometer operating at 300 and 75 MHz respectively. The chemical shift values are reported in  (ppm) relative to SiMe4 (Tetra methyl silane) as an internal standard. The coupling constant (J) is given in Hz.
Plant Material:
The Cassia alata was collected from Karachi (Sindh) and identified by Mr. Ghulam Rasool. A voucher specimen (86464) has been deposited in the herbarium at Department of Botany, Faculty of Science University of Karachi, Sindh Pakistan.
Extraction and Isolation:
The air dried leaves (7 kg), stem (5 kg) and beans (5 kg) of Cassia alata were extracted repeatedly with methanol at room temperature. The solvent was evaporated under vacuum to give 2 kg crude extract of leaves (CA-L), 3 kg crude extract of stem (CA-S) and 750 g crude extract of beans (CA-BN). The dark greenish brown gummy crude extract of beans (CA-BN) was partitioned with ethyl acetate (EtOAc), water (discard) and n-butanol fractions. Each fraction was concentrated in vacuum to have 15 g EtOAc and 15 g n-butanol soluble fractions. The EtOAc soluble fraction was further partitioned with n-hexane to obtained n-hexane soluble fraction and n-hexane insoluble fraction. The n-hexane soluble fraction (14 g) was subjected to vacuum liquid chromatography (VLC) (n-hexane: n-hexane: EtOAc in order of increasing polarity) which furnished 22 fractions (Fr-1-Fr-22). The Fr-15 was subjected to reverse-phase column chromatography using sephadex column LH-20 (CHCl3;CHCl3:MeOH in order of increasing polarity) which yielded 12 fractions (Fr-15-1-Fr-15-12). The Fr-15-9 was further subjected to reverse phase column chromatography using sephadex column LH-20 (n-hexane:CHCl3:MeOH in order of increasing polarity) furnished 18 fractions (Fr-15-9-1-Fr-15-9-18). The Fr-15-9-10 eluted with n-hexane:CHCl3:MeOH (0.5:3:1.5) gave yellow amorphous powder which showed single spot on TLC using CHCl3: MeOH (9.2:0.8) as a solvent system was identified as kaempferol (1) (37 mg). The Fr-15-9-9 was further subjected to reverse-phase column chromatography using sephadex column LH-20 (n-hexane:CHCl3:MeOH in order of increasing polarity) which yielded 13 fractions (Fr-15-9-9-1 to Fr-15-9-9-13). The Fr-15-9-9-3 eluted with n-hexane:CHCl3:MeOH (0.5:3:1.5) showed single spot on TLC (CHCl3:MeOH, 9.2:0.8) appeared as yellowish powder and was identified as luteolin (2) (27 mg).
Biological assay:
Screening of antibacterial activity:
The disc diffusion method (Bauer et al, 1966) was used to determine the antibacterial activity of methanolic extracts. 100 mg/ml of stock solution was prepared by dissolving extracts in DMSO. Sterile filter discs containing 10 l of stock solution were used for screening. The Mueller Hinton agar (Oxoid) plates were seeded with 24 hours old culture grown in Mueller Hinton broth (Oxoid). The prepared discs were placed onto the surfaces at different positions and plates were incubated at 37C for 24 hours. Results were recorded by measuring the zone of inhibitions in mm. Gentamicin was used as a standard.
Screening of antifungal activity:
Antifungal activity was also determined by disc diffusion method (Bauer et al, 1966) as above. Briefly, a small amount of culture was transferred to 2-3 ml distilled water or normal saline in a screw capped tube with few glass beads (1 mm in diameter) and vortexes for 5-10 minutes to make a homogeneous suspension of fungal culture. Sabouraud dextrose agar (SDA) plates were seeded with this suspension. Sterile filter discs containing 10 l of stock solution were placed onto the surfaces at different positions. Plates were incubated at room temperature for 1 week. Results were recorded by measuring the zone of inhibitions in mm. Gresiofulvin was used as a standard.
Determination of minimum inhibitory concentration (MIC):
MIC of methanolic extracts were determined by the disc diffusion method ( Bauer et al, 1966).
Sterile discs containing different concentrations of samples varying from 0.98 to 1000 g per disc were prepared. The MIC of those extracts showing maximum zone of inhibition against microorganism were calculated ( Table 2 ).
Antioxidant activity:
Antioxidant activity was determined by using the method described by Lee et al. (1998). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was prepared in ethanol (300 µM). 10 µL of each extract and 90 μL solution of stable radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added in 96 – well micro titer plates and incubated at 37º C for 30 minutes. Absorbance was measured at 515 nm by using a spectrophotometer. Percent inhibition of radicals by treatment of test sample was determined by comparison with a DMSO treated control group.
% Inhibition = (absorbance of the control-absorbance of the test sample) x 100
Absorbance of the control
Ascorbic acid was used as standard control. The EC50 value calculated denotes the concentration (in ug/ml) of sample required to scavenge 50% of DPPH
Characterization of Kaempferol (1)
Yellow amorphous powder. 1H-NMR  (300 MHz, CD3OD): 8.09 (2H, d, J = 8.7 Hz, H-2’, 6’), 6.91 (2H, d, J = 8.7 Hz, H-3’, 5’), 6.43 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 1.8 Hz, H-6). EIMS m/z: 286 [M]+. 13C-NMR (see Table 1). All data were identical with that of reported in literature (Hadizadeh F., et al, 2003, Gangwal A., et al, 2010).
Characterization of Luteolin (2)
Yellow amorphous powder. 1H- NMR  (300 MHz, CD3OD): 7.39 (1H, dd, J = 9.0, 1.8 Hz, H-6’) ,7.36 (1H, d, J = 1.8 Hz, H-2’), 6.88 (1H, d, J = 9.0, Hz, H-5’), 6.53 (1H, s, H-3), 6.43 (1H, d, J = 1.8 Hz, H-8), 6.19 (1H, d, J = 1.8 Hz, H-6). EIMS m/z: 286 [M]+. 13C-NMR (see Table 1). All data were identical with that of reported in literature (Saeidnia S., et al, 2009).
Results and discussion:
The phytochemical investigation of the methanolic extracts of Cassia alata beans resulted in the isolation of kaempferol (1) and luteolin (2). Compound (1) showed molecular ion peak at m/z 286 having molecular formula C15H10O6. Its 1H-NMR spectrum showed the characterstic peak of H-2’ and 6’ as a doublet at with ortho coupling of 8.7 Hz whereas H-3’ and 5’ with similar ortho coupling appeared at 6.91 as a doublet.1H-NMR spectrum of 1 further displayed signals of aromatic protons as a doublet at 6.19 (H-6) and at  6.43 (H-8) showing meta coupling of 1.8 Hz.
The EIMS spectrum of compound (2) is similar to (1) having same molecular mass (m/z 286) and formula (C15H10O6). In the 1H-NMR spectrum of 2 characterstic peak of H-3 appeared at 6.53 as a singlet. Other important signals observed at 6.88 (d, J = 9.0, Hz, H-5’), 7.36 (d, J = 1.8 Hz, H-2’) and 7.39 (dd, J = 9.0, 1.8 Hz, H-6’). The aromatic protons H-6 and H-8 showed same and J value as in compound 1. The 13C-NMR spectrum (Table 1) of both compounds 1 and 2 displayed signals of nine quaternary and six methine carbons . All the 13C assignments are in agreement with the reported data (Gangwal A., et al, 2010, Saeidnia S., et al, 2009).
The results of antibacterial activity indicated that all the methanolic extracts of C. alata (CA-L, CA-S and CA-BN) have potential to kill various pathogenic gram+ve and gram-ve bacteria (Table 2), whereas good antifungal activity was observed in CA-S and CA-BN extracts against Fusarium specie (Table 3). All the extracts (CA-L, CA-S and CA-BN) showed less than 50% inhibition of DPPH radicals in antioxidant activity (Table 4).
CONCLUSION:
The known flavonoids kaemferol (1) and luteolin (2) were isolated from the methanolic extracts of C. alata beans. The structure of the isolated compounds were elucidated by various spectroscopic techniques. Pharmacological investigations have indicated that all the extracts (CA-L, CA-S and CA-BN) of this plant possess significant antimicrobial and weak antioxidant activity.
References:
Bauer, A.W., Kirby, W.M.M., Sherris, J.C., & Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45, 493–496.
Bhattarchrjee, S.K. and Michael,A.M. (2004). Hand Book of Medicinal Plants. Pointer Publishers Jaipur 302003 (Raj), India, pp. 77-78.
Gangwal, A., Parmar, S.K., Sheth, N.R. (2010). Vol. 2(1). Triterpenoid, flavonoids and sterols from Lagenaria siceraria fruits. Scholars Research Library. pp. 307- 317.
Hadizadeh, F., Noaman, K., Hossein, H., Randa, K.A. (2003). Kaempferol from Saffron Petals. Iranian Journal of Pharmaceutical Research. pp. 251-252.
Joshi, S.G. (2000). Medicinal Plants. Oxford and IBH publishing Co.Pvt. Ltd, New Delhi, Calcutta, India, pp. 117.
Khare, C.P. (2007). Indian Medicinal Plants.Springer.New Delhi, India, pp.126.
Krishnan, M.K.S. (1992). Vol.3. The Wealth of India. Council of Scientific and Industrial Research New Delhi, India, pp. 328.
Lee, S. K., Zakaria, H., Chuyng, H. L., Kuyengl, L.,Games, E. J. C., Mehta, R. J., Kinghorn, D., and Pezzuto, J. M. (1998). Evaluation of the antioxidant potential of natural products.Combinatorial Chemistry and High Throughput Screening.1: 35-4
Neharkar, V.S., Gaikwad, K.G. (2011). Vol. 2(1). Hepatoprotective activity of Cassia alata (Linn.) leaves against Paracetamol-induced hepatic injury in rats. Research Journal of Pharmaceutical, Biological and Chemical Sciences: pp. 783-788.
Quattrocchi, U., F.L.S. (2012). Vol.5 R-Z: CRC World Dictionary of Medicinal and Poisonous Plants. CRC Press Taylor & Francis Group Boca Raton New York, USA, pp. 236-237.
Ray, A.B., Chansouria, J.P.N. and Hemalatha, S. (2010). Medicinal Plants: antidiabetic and Hypoglycaemic Activities. ibdc Publishers Lucknow, India, pp. 95.
Ross, I.A. (2003). vol.1. Medicinal Plants of the World. Humana Press, Totowa, New Jersey, pp. 165-166.
Reezal, I., Somchit, M. N. and Abdul Rahim, M. (2002). Vol.1. In vitro Antifungal Properties of Cassia alata (GELNGGANG BESAR). Proceedings of the Regional Symposium on Environment and Natural Resources. pp. 654-659.
Saeidnia, S., Yassa, N., Rezaeipoor ,R., Shafiee, A., Gohari, A. R., Kamalinejad, M., Gooderzy, S. (2009). Vol. 17(1). Immunosuppressive principles from Achillea talagonica, an endemic species of Iran. Journals.tums.ac.ir: pp. 37-41.

Table 1. C13-NMR spectral data of kaempferol (1) and luteolin (2) in CD3OD (ppm) at
75 MHz

Carbon

1

2

2

146.3

164.3

3

135.2

103.2

4

175.2

181.7

5

160.4

161.5

6

98.4

99.1

7

163.7

164.3

8

93.8

94.2

9

156.7

157.6

10

103.1

104.1

1`

122.1

122.0

2`

129.4

112.8

3`

115.3

146.1

4`

158.7

150.0

5`

115.3

115.3

6`

129.4

119.2

Table 2. Antibacterial activity of different extracts of Cassia alata (zone of inhibition
in mm)

Gram positive
bacteria

Zone of inhibition in mm

MIC
(µg/disc)

CA-L

CA-S

CA-BN

Standard
Gentamicin

CA-L

CA-S

CA-BN

Bacillus cereus

–

–

–

>15

–

–

–

Bacillus subtilis

9

–

–

>15

–

–

–

Bacillus thereugenesis

–

–

–

>15

–

–

–

Corynebacterium diphtheriae

11

–

–

>15

–

–

–

Corynebacterium hofmanii

–

–

–

>15

–

–

–

Corynebacterium xerosis

–

–

–

>15

–

–

–

Micrococcus leutus ATCC9341

–

–

–

>15

–

–

–

Micrococcus leutus

–

–

–

>15

–

–

–

Streptococcus fecalis

–

–

–

>15

–

–

–

Streptococcus pyogenes
Streptococcus saprophyticus

11
–

9
–

9
–

>15
>15

–
–

–
–

–
–

Methicillin resistant
staphylococcus aureus

10

11

12

>15

–

–

–

Staphylococcus aureus

–

–

–

>15

–

–

–

Staphylococcus aureus
AB188
Staphylococcus epidermidis

–
13

11
13

14
14

>15
>15

–
–

–
–

–
–

 
 
 
 
 
 
 
 

Gram negative
bacteria

 
 
 
 
 
 
 

Enterobacter

15

14

15

>15

500

–

–

Escherichia coli ATCC 8739

–

–

–

>15

–

–

–

Escherichia coli

–

–

–

>15

–

–

–

E. coli multi drug resistance

–

–

–

>15

–

–

–

Klebsiella pneumonae

24

23

26

>15

250

250

250

Proteus mirabilis

–

–

–

>15

–

–

–

Pseudomonas aeroginosa ATCC

12

14

14

>15

–

–

–

Salmonella typhi

12

–

13

>15

–

–

–

Salmonella paratyphi A

–

–

–

>15

–

–

–

Salmonella paratyphi B

–

–

–

>15

–

–

–

Shigella dysentery

13

13

14

>15

–

–

–

Shigella flexenerii

–

–

–

>15

–

–

–

Pseudomonas aeroginosa

–

–

–

>15

–

–

–

 
 
 
 
 
 
 
 
 

CA-L= Cassia alata Leaves, CA-S = Cassia alata Stem, CA-BN = Cassia alata Beans.
 
Table 3. In Vitro Antifungal activity (zone of inhibition in mm)

YEAST

Zone of inhibition in mm
(mean+S.D)

 

CA-L

CA-S

CA-BN

Standard
Gresiofulvin

Candida albicans

–

–

–

>12

Candida albicans ATCC 0383

–

–

–

>12

Saccharomyces cerevisiae

–

–

–

>12

DERMATOPHYTES

 
 
 
 

Microsporum canis

–

–

–

>12

Microsporum gypseum

–

–

–

>12

Trichophyton rubrum

–

–

–

>12

Trichophyton tonsurans

–

–

–

>12

Trichophyton mentagrophytes

–

–

–

>12

 
 
 
 
 

SAPROPHYTES

 
 
 
 

Aspergillus flavus

–

–

–

>12

Aspergillus niger

–

–

–

>12

Fusarium specie

–

13

13

>12

Penicillium specie

–

–

–

>12

Rhizopus

–

–

–

>12

Helminthosporum

–

–

–

>12

 
 
 
 
 

Table 4. Antioxidant activity of Methanolic extracts of C.alata

S. NO.

SAMPLES

% Inhibition

EC50 ug / ml

01

CA-L

38.2

–

02

CA-S

40.11

–

03

CA-BN

22.52

–

04

Ascorbic acid

80%

–