1. INTRODUCTION:

Since the advent of modern drug treatments, traditional medicine has greatly receded in occidental societies. Moreover, only a limited number of medicinal plants have received detailed scientific scrutiny thereby prompting the World Health Organization to recommend that this area be comprehensively investigated²³. Cassia pumila is used extensively in various parts of the world against a wide range of ailments, the synergistic action of its metabolite production being most probably responsible for the plant’s beneficial effects^18,25. Plants used as source of indigenous medicines due presence of various secondary metabolites. In the present investigation, the flavonoids with a new flavonoid were isolated and their antimicrobial activities were carried out. Among natural phenolics, the flavonoid forms the largest group and more than 2,000 flavonoids are reported among woody and non-woody plants ^{8,9,14, 35}.

A number of plants have been screened for the presence of flavonoids, by many workers such as Hahlbrock and Risebach, 1975 ⁷; Wassel and Baghdadi, 1979 ³⁴; Takahashi et al., 1981²⁸; Markus et al., 2008¹⁵. Thus traditional medicinal plants derived antioxidants may protect against a number of diseases and reduce oxidation processes in food systems ^10,20. In order to establish this, it is imperative to measure the markers of baseline oxidative stress particularly in human health and disease and examine how they are affected by supplementation with pure compounds or complex plant extracts from the traditional medicinal plants¹¹. The data so far generated clearly sets the basis for a clear understanding of the phytochemistry of the plant and derived cultures and opens the possibility of the potential utilization of the phenolic rich extracts from medicinal plants in food system or as prophylactics in nutritional/food supplement programs^2,20. The flavonoids have utmost importance in providing strength and resistance to the plant against various diseases but also exhibit various biological and pharmacological activities^{19, 23, 3, 32}. A survey of literature suggested that the flavonoids are found both, in free and bound forms, the former being in higher levels than the later³¹.

In the present study a new flavonoid kampferol-7-O-glucoside with the other flavonoids were isolated by chromatography and characterized them by spectral studies and antimicrobial activity of these isolated compounds was carried out by following the established protocols.

2. MATERIAL AND METHODS:

2.1Collection and Identification

Cassia pumila is a ornamental tree which is found usually in the shade of trees, crevices of rocks and also in the open gravelly substratum, often hidden amongst grasses during rainy season at surroundings of Jai Gragh fort in Jaipur. The plant was identified at Herbarium, Department of Botany, University of Rajasthan, Jaipur, and their voucher specimen (No. 3026) has been deposited in the Harbarium.

2.2 Processing and Extraction

The plant parts (root, stem, leaves, flowers and pods) of C. pumila were studied for flavonoids composition. Plant sample was extracted with 80% methanol²⁷ and the concentrated extract was fractionated with pet. ether (Fr. I). diethyl ether (Fr. II) and ethyl acetate (Fr. III) successively. Fr. I was however, rejected in each case being rich in fatty components whereas, Fr. II and Fr. III were analysed for free and bound flavonoids respectively. Later, Fr. III was acid-hydrolysed (7% H₂SO4; 10 ml/g) for 2 hand re-extracted with ethyl acetate (Fr. IV), followed by neutralization ³⁴. Both the fractions (II and IV) were co-chromatographed on tlc (Silica gel; Solvent: benzene - acetlc acid - water, 125: 72: 3) along with the standard markers³⁵. The developed chromatograms were viewed under UV light alone and in the presence of ammonia fumes and subsequently sprayed with the characteristic reagents¹⁷.Three fluorescent spots coinciding to reference kaempferol (R_f 0.85), quercetin (R_f 0.78), and kaempferol-7-O-glucoside(R_f 0.81) were isolated by tlc eluted and purified. Later, the isolated compounds were crystallized and identified by using MP, UV, IR and NMR spectroscopy (Veitech and Grover, 2008). Using spectrophotometric methods of Mabry et al. (1970)¹⁴ and Kariyone et al. (1953)¹², the quantification was made of kaempferol, quercetin and kaempferol-7-O-glucoside respectively.

2.3 Test Microorganisms

Standard strains of Staphylococcus aureus, Salmonella typhyi, Escherichia coli and Pseudomonas aeruginosa were obtain from microbiology Lab. SMS medical college, Jaipur, Rajasthan and Aspergillus flavus, A. niger, Fusarium monilliformae and Rhizoctonia bataticola were obtain from seed pathology lab, Department of Botany, University of Rajasthan, Jaipur, India.

2.4 Antimicrobial Screening

The Disc Diffusion method was used to determine the antimicrobial activities of the isolated flavonoid using standard procedure using standard procedure⁴ of 6 mm disc were prepared from whatman’s filter paper no. 1. Solutions of varying concentrations ranging from 1.0 × 10⁴ to 5.0 × 10⁴ mg/ml/disc were prepared. They were also prepared using the pure extruding solvent for each extract. Nutrient agar was prepared, sterilized and used as the growth medium for the culture of microorganisms, 20ml of the sterilized medium was poured into each sterilized petri dish, covered and allowed to solidify. The treated discs were air dried at room temperature, to remove any residual solvent which might interfere with the determination, sterilized and inoculated.

These plates were initially placed at low temperature for 1 hour so as to allow the maximum diffusion of compounds from the test disc into the agar plate and later incubated at 37°C for 24 hours in case bacteria and 48 hours for fungi, after which the zone of inhibition could be easily observed five replicates of each text extract were examined and the mean values were then referred.

3. RESULT AND DISCUSSION:

3.1 Structure Elucidation of Isolated Flavonoids

In the present investigation, flavonoids profile has been studied in vivo of C. pumila, where kaempferol-7-O-glucoside, kaempferol and quercetin from different plant parts of C. pumila have been evaluated by chromatographic, spectroscopic and color reactions. The compounds eluted from tlc were pooled together according to their tlc behaviour and isolate them with the solvents and evaporated yielding three flavonoids kaempferol-7-O-glucoside, kaempferol and quercetin³². The spectral analyses of the active constituent, Spectra-I - kaempferol-7-O-glucoside, Spectra-II - kaempferol and Spectra-III - quercetin from the different plant parts of selected C. pumila are shown below: -

Spectra-Ia-c: Kaempferol-7-O-glucoside: brownish needles on crystallization (mp 317°-329°C)

UV light absorption MeOH: 235 sh, 240 sh, 259 sh, 374 sh, 424 sh

IR : vcm^–1/ max KBr: 3600 (glycoside), 3420 (O–H), 1700 (C=O), 1600, 1610, 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010, 815, Spectrum-I a

¹HNMR(300MHz, CDCl₃): 5.1 (H₁), 6.68 (H₂), 7.64 (H₃), 6.04 (H₄), 6.03 (H₅), 5.20 (H₆), 6.81 (H₇), 7.16 (H₈), 6.70 (H₉), 5.91 (H₁₀), 3.91 (H₁₁), 2.37 (H₁₂), 3.40 (H₁₃), 2.48 (H₁₄), 3.76 (H₁₅), 2.41 (H₁₆), 3.49 (H₁₇), 2.31 (H₁₈), Spectrum-I b

¹³C NMR(300MHz, CDCl₃): 70.6 (C₁), 75.3 (C₂), 78.8 (C₃), 92.4 (C₄), 154.8 (C₅), 154.2 (C₆), 114.6 (C₇), 137.5 (C₈), 124.0 (C₉), 136.0 (C₁₀), 121.1 (C₁₁), 149.4 (C₁₂), 97.5 (C₁₃), 123.1 (C₁₄), 129.0 (C₁₅), Spectrum-I c

Spectra-IIa-c: Kaempferol: brownish needles on crystallization (mp 312°-313°C)

UV light absorption MeOH: 253 sh, 269 sh, 305 sh, 374 sh, 424 sh

IR: vcm^–1/ max KBr: 3420 (O–H), 2830 (C-H), 1700 (C=O), 1600, 1610, 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010, 815, Spectrum-IIa

¹HNMR(300MHz, CDCl₃): 2.35(H₁), 7.01(H₂), 7.18 (H₃), 6.29 (H₄), 6.37 (H₅), 2.35 (H₆), 5.39 (H₇), 5.36 (H₈), 7.18 (H₉), 7.01 (H₁₀), Spectrum-IIb

(c)¹³C NMR(300MHz, CDCl₃): 1.36 (C₁), 129.8 (C₂), 126.8 (C₃), 131.9 (C₄), 147.4 (C₅), 154.2 (C₆), 114.6 (C₇), 137.5 (C₈), 124.0 (C₉), 136.0 (C₁₀), 121.1 (C₁₁), 149.4 (C₁₂), 106.9 (C₁₃), 131.9 (C₁₄), 126.1 (C₁₅), Spectrum-IIc

Spectra-IIIa-c: Quercetin: yellowish needles on crystallization (mp 312°-313°C)

UV light absorption MeOH: 255 sh, 301 sh, 374 sh, 440 sh

IR : vcm^–1/ max KBr: 3420,3380(O–H), 2800 (C-H), 1680 (C=O), 1610, 1610, 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010, Spectrum IIIa.

¹HNMR (300MHz, CDCl₃): 2.45, (H₁), 2.55 (H₂), 6.79 (H₃), 6.98 (H₄), 6.49 (H₅), 2.33 (H₆), 6.38 (H₇), 2.36 (H₈), 5.37 (H₉), 1.4 (H₁₀), Spectrum IIIb.

¹³C NMR (300MHz, CDCl₃): 137.3 (C₁), 137.9 (C₂), 14.2 (C₃), 127.0 (C₄), 126.1 (C₅), 133.8 (C₆), 142.4 (C₇), 158.2 (C₈), 114.6(C₉),134.5 (C₁₀), 123.0 (C₁₁), 138.0 (C₁₂), 121.1 (C₁₃), 149.4 (C₁₄), 108.9 (C₁₅), 127.8, Fig. IX.

3.2Concentration of isolated flavonoids

The results of the chromatographic data and concentration of isolated flavonoids contents (mg/gdw) are summarized in table 1 and 2 respectively. The isolated flavonoids were identified on the basis of color reactions UV light, I₂ vapours, spraying with chromogenic reagents and by the R_f values as mentioned in table 1.

Although both free and bound flavonoids were isolated from all the plant part but the bound form of newly identified flavonoid- kaempferol-7-O-glucoside was found absent in roots, stem and pods. The maximum concentration of both free and bound flavanoids was observed in the flowers (free = 0.28 mg/gdw, bound = 0.32 mg/gdw). The minimum concentration of both free and bound flavonoids was observed in flowers i.e. 1.92 mg/gdw and 0.10 mg/gdw respectively. Among the isolated flavonoids, kaempferol was recorded in more concentration in flower (0.62 mg/gdw, free form) than the isolated other flavonoids. In the bound form kaempferol was recoded in higher concentration (0.16 mg/gdw) in flowers also. Over all in the whole plant the isolated free flavonoids were recorded in higher concentration (1.62 mg/gdw) than the bound flavonoids (0.32 mg/gdw).

Table 1 : Chromatographic data and colour reaction of the flavonoids isolated from C. pumila.

Flavonoids (aglycones)	Rf(×100) in					Colours by chromatogenic sprays colour
	BeAW⁺	BAW^*	TBA⁺⁺	Day- light	*UV^ ammonia**	I₂ vapours	FeCl₃		AlCl₃
	BeAW⁺	BAW^*	TBA⁺⁺	Day- light	*UV^ ammonia**	I₂ vapours	Visible	UV	Visible
Kaempferol-7-O-glucoside	81	78	49	YN	BN	BN	BN	BW	YW	YW
Kaempferol	85	83	55	GN-YW	BT-YW	YW-BN	BN	BK	YW	YW-GN
Quercetin	78	64	41	GN-YW	YW	YW-BN	BT-GY	BK	DL-YW	YW-GN

Abbreviations : ⁺BeAW = Benzene : Acetic acid : Water (125 : 72 : 3); BK = Black; BN = Brown; BT = bright

*BAW = n-Butanol : Acetic acid : Water : (4: 1:5); DL = dull; GN = green; YW = yellow

⁺⁺TBA = t-Butanol : Acetic acid : Water (3:1:1)

Table 2 : Isolated flavonoid content (mg/gdw*) in the Cassia pumila.

Plant species	Free (F)
Plant species	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total
C. pumila
Root	0.17	0.12	0.10	0.39
Stem	0.18	0.10	0.15	0.43
Leaves	0.45	0.35	0.23	1.03
Flowers	0.86	0.46	0.28	1.60
Pods	0.56	0.43	0.14	1.13
Total	0.17	0.12	0.10	0.39

Plant species	Bound (B)
Plant species	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total
C. pumila
Root	0.09	0.07	0.04	0.20
Stem	0.06	0.04	0.05	0.15
Leaves	0.12	0.10	0.06	0.28
Flowers	0.11	0.16	0.05	0.32
Pods	0.10	0.16	0.04	0.30
Total	0.09	0.07	0.04	0.20

Plant species	Total (F+B)
Plant species	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total
C. pumila
Root	0.26	0.19	0.14	0.59
Stem	0.24	0.14	0.20	0.58
Leaves	0.57	0.45	0.29	1.31
Flowers	0.97	0.62	0.33	1.92
Pods	0.66	0.59	0.18	1.53
Total	0.26	0.19	0.14	0.59

Table 3 : Bactericidal and fungicidal efficacy of isolated flavonoids from Cassia pumila.

Test organist		Flavonoids
Test organist		Kaempferol	Quercetin	Kaempferol-7-O-glucoside
A. Bacteria
E. coli	IZ^*	15.00	26.00	17.00
E. coli	AI^*	0.62	1.02	0.68
S. aureus	IZ^*	12.00	11.00	15.00
S. aureus	AI^*	0.50	0.45	0.62
P. aeruginosa	IZ^*	11.0	12.00	7.00
P. aeruginosa	AI^*	0.41	0.46	0.28
S. typhi	IZ^*	9.00	11.00	12.00
S. typhi	AI^*	0.31	0.45	0.50
B. Fungi
A. flavus	IZ^*	22.00	24.00	16.00
A. flavus	AI^*	0.85	0.74	0.80
A.niger	IZ^*	21.00	24.00	9.00
A.niger	AI^*	0.85	0.74	0.56
R. bataticola	IZ^*	6.00	18.00	21.00
R. bataticola	AI^*	0.20	0.73	0.84
F. moniliforme	IZ^*	6.00	8.00	16.00
F. moniliforme	AI^*	0.33	0.38	0.76

(+) Trace activity, *IZ = Inhibition zone (in mm) including the diameter of disc (6 mm) (-) Not measurable activity

Standard : Zentamycin = 10µg/disc

3.3 Antimicrobial activity:

Histochemically plant have provided good source of anti-infective agents which are highly effective against the microbes. The present study was aimed at determining the phytochemistry and antimicrobial activities of isolated flavonoids against pathogenic bacteria and fungi Table 3 and plate-1.

The results of antibacterial activity showed that all the isolated flavonoids were effective against all test bacteria but quercetin was recorded more effective against E. coli (IZ= 26 mm) while kaempferol-7-O-glucoside was very less effective against P. aeuroginosa (IZ = 7 mm) but it is more effective against the S. typhi (IZ = 12 mm) in comparison to other isolated flavanoids. The antifungal activity was carried out against the test fungi. All the test fungi were found sensitive to the isolate flavonoids but again quercetin was found more effective against A. flavus (IZ = 24 mm) and A. niger (IZ = 24 mm) and minimum inhibition activity was showed by kaempferol against R. bataticola (IZ = 6 mm) and F. moniliformae (IZ = 6 mm).

Table 4 shows the results of MIC for isolated flavonoids for against test microorganisms recorded in mg/disc of the diametrical sections of the respective zones of inhibition for each metabolite. The quercetin was higher effective against E. coli, A. flavus, A. niger, F. moniliformae and R. bataticola therefore the MIC value 2×10³ mg/disc was recorded and MIC value for other bacteria and fungi 3×10³ mg/disc was recorded. Kaempferol was more active only A. flavus, A. niger, F. moniliformae and R. bataticola therefore the MIC value 2×10³ mg/disc was recorded. Kaempferol-7-O-glucoside shows MIC value 2×10³ mg/disc for E. coli, A. flavus and A. niger but MIC value for S. aureus, P. aeurinosa and S. typhi 3×10³ mg/disc was recorded.In general, work on the flavonoid, in Cassia species has centred on the chemical aspects and accordingly a number of glycosides have been characterized, viz. : Kaempferol-3-O--D-monopyranosyl from C. grantis and C. auriculata^21,26, 3,5,3´,4´,5´-pentahydroxy-7-methoxyflavone-8-c-l-rhamnopyranoside from C. sophera³⁰, Velutin (5,4´-tihydroxy-7,3´-dimethoxy flavone from C. pumila²⁹, 5,7,3,5´-tetrahydroxy-6,8, dimethoxy flavone-3-o-µ-arobinopyranosite and 5,7,4´-trihydroxy-6,8,3´-trimethoxy flavone-3-O-µ-L-rhamnosyl (12)-O--D-gluco-pyranoside from C. fistula^5,6, apigenin 6-c--D-olioside from C. torosa¹³ and some aglycones- kaempferol, quercetin and myricetin from C. biflora ^1,16,17, Kaempferol-7-methylether from C. javanica ²².

Some species of the genus Cassia exhibited pathological-physiological and biological-activities, their chemistry has been extensively studied, incidentally, very less attention has been paid to study the flavonoid pattern in general, and the Cassia species, in particular, but very few reports have been published on the production of flavonoids and their antimicrobial activities in Cassia species ^{1,16,17,24,26,29,30}.

Table 4 : Zones of inhibition of different concentration (MIC) of isolated flavonoids (mg/ml).

Test microorganism

Quercetin

Kaempferol

Kaempferol- 7-O-glucoside

1×10³

2×10³

3×10³

4×10³

5×10³

1×10³

2×10³

3×10³

4×10³

5×10³

1×10³

2×10³

3×10³

4×10³

5×10³

Bacteria

E. coli

S. aureus

P. aeurginosa

S. typhi

Fungi

A. flavus

A. niger

R.. bataticola

F. moniliformae

MIC = Minimum inhibitory concentration

Plate-1 : Photographs of antimicrobial activity of isolated flavonoids from selected Cassia species.

Bacteria

A = E. coliB = S. aureus

C = P. aeruginosa D = S. typhi

Fungi

E = A. flavus F = A. niger

G = R. bataticola H = F. moniliforme

1 = Kaempferol

2 = Quercetin

3 = Kaempferol-7-O-glucoside

S = Standard gentamycin for bacteria

S = Standard mycostatin for fungi

On the basis of above results, it is concluded that the C. pumila accumulates certain secondary metabolites in different parts which shows various antimicrobial and pharmacological activities. Therefore, it is a potential source of indigenous medicine to cure various ailments.

4. Reference:

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7) Hahlbrock K, Risebach HG. 1975. Biosynthesis of flavonoids. In: The Flavonoids. Harborne JB, Mabry TJ, Mabry H (eds.), Chapman and Hall, London, pp. 866-915.

8) Harborne JB. 1964. Phenolic glycosides and their natural distribution. In: Biochemistry of Phenolic Compounds. Harborne JB, Academic Press, London, , pp. 129-170.

9) Harborne JB. 1967. Comparative Biochemistry of the Flavonoids. Academic Press, London.

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14) Mabry TJ, Markham KR, Thomas MB. 1970. The Systematic Identification of Flavonoids, Springer-Verlag, Berlin.

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Received on 21.12.2011 Modified on 15.01.2012

Asian J. Research Chem. 5(2): February 2012; Page 305-311

2.3 Test Microorganisms

Colours by chromatogenic sprays colour

Flavonoids

Quercetin

Kaempferol-7-O-glucoside

A. Bacteria

15.00

0.62

7.00

0.28

12.00

0.50

B. Fungi

16.00

0.80

9.00

0.56

21.00

0.84

16.00

0.76

Bacteria

Fungi

Bacteria

Fungi

S = Standard gentamycin for bacteria

S = Standard mycostatin for fungi