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 Organisation to recommend that this area should be comprehensively investigated. Cassia species are 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 plants beneficial effects^1-2. The present investigation, the primary and secondary metabolites composition of vegetative and reproductive plant parts thereby derived, with emphasis on potent phenolic antioxidants such as, flavonoids.

Among natural phenolics, the flavonoid forms the largest group and more than 2,000 flavonoids have been reported among woody and non-woody plants³. The occurrence of free aglycones, however, appears to be mostly correlated with the existence of secretory structures. The flavonoids have more importance in providing strength and resistance to the plant against various diseases but also exhibit various biological and pharmacological activities.

A survey of literature suggested that the flavonoids are found both, in free and bound forms, the former being in higher levels than the latter.

In general, work on the flavonoids in Cassia species has centred on the chemical aspects and accordingly a number of glycosides have been characterized, viz. : kaempferol-3-O-β-D-monopyranosyl from Cassia grandis and Cassia auriculata^4-5; 3, 5, 3’, 4’, 5’ – pentahydroxy-7-methoxyflavone-8-C-l-rhamnopyranoside from Cassia sophera⁶; velutin (5,4’-dihydroxy-7,3’-dimethoxy flavone) from Cassia nodosa⁷; 5, 7, 3, 5’ – tetrahydroxy-6, 8, dimethoxy flavone-3-O-∞-arabinopyranoside and 5, 7, 4’-trihydroxy-6, 8, 3’-trimethoxy flavone-3-O-∞-L-rhamnosyl (1→2)-O-β-D-gluco-pyranoside from Cassia fistula^8-9; apigenin 6-C-β-D-olioside and diosmethin G-C-β-D-olioside from Cassia torosa¹⁰; some aglycones - kaempferol, quercetin and myricetin from Cassia biflora ^11-13 and kaempferol-7-methylether from Cassia javanica¹⁴.

MATERIAL AND METHODS:

Collection of Plant material:

Cassia nodosa Bunch. and C. renigera Wall. the ornamental trees while the C. pumila is weed of rainy season. The plant material of C. nodosa and C. renigera were collected from the Central Park of Jaipur and Kapoor Chand Koolish Garden, Jawahar Lal Nehru Marg, Jaipur. While C. pumila was collected from Jaygarh fort Amber, Jaipur and Garganesh temple of Jaipur.

Identification of Plant material:

All the three plant species were authenticated at source and voucher specimens have been deposited in the Herbarium of the Department of Botany, University of Rajasthan. Jaipur.

Processing of plant materials:

Each of the plant materials was washed in water, shade dried and milled before use during the course of the study.

Extraction procedure of flavonoids:

The all plant parts (root, stem, leaves, flowers and pods) of three Cassia species were studied for flavonoid contents. Each plant sample was extracted with 80% methanol ¹⁵ and the concentrated extract was fractionated with pet. ether (Fraction I). diethyl ether (Fraction II) and ethyl acetate (Fraction 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 hydrolyzed by acid (7% H₂SO4; 10 ml/g) and re-extracted with ethyl acetate (Fr. IV), followed by neutralization. Thin layer chromatography of Fr. II and Fr. IV were done along with the standard markers. All the solvents were used of analytical grade from Merck (India). The TLC Aluminum sheet, 60 F₂₅₄ (20×10 cm) (Cat. No. 1.05554.0007) was purchased from E. Merck (Mumbai). A number of solvent tried for good resolution but better resolution has been found solvent system (benzene: acetic acid: water: 125 : 72 : 3). The developed chromatograms were viewed under UV light alone and in the presence of ammonia fumes and subsequently sprayed with the characteristic reagents.

Fluorescent spots of the samples coinciding with the standard. Bands are matched with the standard at kaempferol (R_f 0.85), quercetin (R_f 0.78), and kaempferol-7-O-glucoside (R_f 0.81). These samples were isolated by TLC; then it is eluted and purified. Later, the isolated compounds were crystallized and identified by using melting point, UV, IR and NMR spectroscopy. Using spectrophotometric methods of the quantification was made of kaempferol, quercetin and kaempferol-7-O-glucoside, respectively^16-17.

RESULT AND DISCUSSION:

In the present investigation, flavonoids profile has been studied in vivo of the selected Cassia species, where quercetin, kaempferol and kaempferol-7-O-glycoside from different plant parts of C. nodosa, C. renigera and C. pumila have been evaluated, their chromatographic, spectroscopic and color reactions data have been presented in the Table-1.

The eluted compounds from TLC were pooled together according to their TLC behaviour and isolate them with the suitable solvents and evaporated yielding three flavonoids kaempferol, quercetin and kaempferol-7-O-glycoside. The spectral analyses of the active constituent, (a) kaempferol (b) quercetin and (c) kaempferol-7-O-glucoside from the different plant parts of selected Cassia species are shown below: -

(a) Kaempferol (Fig.-1): brownish needles on crystallization (m.p. 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), 2240 (C=C), 1700 (C=O), 1600, 1610 (C≡C), 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010, 815;

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);

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).

Figure 1: Structure of Kaempferol

(b) Quercetin (Fig.-2): yellowish needles on crystallization (m.p. 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), 2100 (C=C), 1680 (C=O), 1610 (C≡C), 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010;

¹H NMR (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);

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.

Figure 2: Structure of Quercetin

Table 1 : Isolated flavonoid content (mg/gdw) in the selected Cassia species.

Plant species		Free (F)				Bound (B)				Total (F+B)
Plant species		Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total
1.	C. nodosa
	Root	0.04	0.06	0.00	0.10	0.03	0.10	0.00	0.13	0.07	0.16	0.00	0.23
	Stem	0.02	0.09	0.02	0.13	0.04	0.00	0.00	0.04	0.06	0.09	0.02	0.17
	Leaves	0.10	0.35	0.06	0.51	0.13	0.06	0.00	0.19	0.23	0.41	0.06	0.70
	Flowers	0.85	0.55	0.26	1.66	0.17	0.38	0.16	0.71	1.02	0.93	0.42	2.37
	Pods	0.10	0.15	0.03	0.28	0.05	0.03	0.00	0.08	0.15	0.18	0.03	0.36
2.	C. renigera
	Root	0.05	0.04	0.00	0.09	0.02	0.03	0.00	0.05	0.07	0.07	0.00	0.14
	Stem	0.04	0.05	0.00	0.09	0.03	0.02	0.00	0.05	0.07	0.07	0.00	0.14
	Leaves	0.12	0.33	0.08	0.53	0.17	0.04	0.02	0.23	0.29	0.37	0.10	0.76
	Flowers	0.55	0.40	0.25	1.20	0.16	0.20	0.10	0.46	0.71	0.60	0.35	1.66
	Pods	0.17	0.18	0.04	0.39	0.06	0.05	0.02	0.13	0.23	0.23	0.06	0.52
3.	C. pumila
	Root	0.02	0.07	0.02	0.11	0.02	0.00	0.00	0.02	0.04	0.07	0.02	0.13
	Stem	0.04	0.08	0.03	0.15	0.04	0.02	0.00	0.06	0.08	0.10	0.03	0.21
	Leaves	0.10	0.25	0.04	0.39	0.09	0.04	0.02	0.15	0.19	0.29	0.06	0.54
	Flowers	0.24	0.46	0.16	0.86	0.10	0.14	0.05	0.29	0.34	0.60	0.21	1.15
	Pods	0.13	0.16	0.04	0.33	0.06	0.03	0.00	0.09	0.19	0.19	0.04	0.42

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

IR : vcm^–1/ max KBr: 3600 (glycoside), 3420 (O–H), 2210 (C≡C), 1700 (C=O), 1600 (C=C), 1610, 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010, 815;

¹H NMR (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);

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).

Figure 3: Structure of Kaempferol-7-O-glucoside

The higher levels of total flavonoids (F + B form), following the other selected Cassia species were found in the flowers of (C. nodosa; 2.37 mg/gdw > C. renigera; 1.66 mg/gdw > C. pumila; 1.15 mg/gdw) and minimum concentration was measured in roots (C. pumila; 0.04 mg/gdw < C. renigera; 0.07 mg/gdw) but in C. nodosa minimum concentration was measured in stem (0.17 mg/gdw). The maximum levels of free form of flavonoid was also observed in flowers (C. nodosa; 1.56 mg/gdw > C. renigera; 1.20 mg/gdw > C. pumila; 0.86 mg/gdw) and likewise minimum levels was measured in roots (C. renigera; 0.09 mg/gdw < C. nodosa; 0.10 mg/gdw < C. pumila; 0.11 mg/gdw). Similarly the higher concentration of total bound flavonoids in flowers (C. nodosa; 0.71 mg/gdw > C. renigera; 0.46 mg/gdw > C. pumila; 0.19 mg/gdw) and minimum levels was also observed in roots (C. pumila; 0.02 mg/gdw < C. renigera; 0.05 mg/gdw), but in C. nodosa minimum concentration was measured in stem (0.04 mg/gdw, Table-1).

The higher concentration of free form of quercetin was found in flowers (C. nodosa; 0.55 mg/gdw > C. pumila; 0.46 mg/gdw > C. renigera; 0.40 mg/gdw) and minimum concentration was measured in roots
(C. renigera; 0.04 mg/gdw < C. nodosa; 0.06 mg/gdw < C. pumila; 0.07 mg/gdw), and higher concentration of quercetin (bound form) was measured in flowers (C. nodosa; 0.17 mg/gdw > C. renigera; 0.16 mg/gdw >
C. pumila; 0.10 mg/gdw) and minimum concentration was measured in roots (C. pumila; 0.02 mg/gdw = C. renigera; 0.02 mg/gdw < C. nodosa; 0.03 mg/gdw, Table-1).

The concentration of free form of kaempferol was measured in flowers (C. nodosa; 0.85 mg/gdw > C. renigera; 0.55 mg/gdw > C. pumila; 0.24 mg/gdw) and minimum levels was measured in roots (C. pumila; 0.02 mg/gdw < C. nodosa; 0.04 mg/gdw < C. renigera; 0.05 mg/gdw). Similarly higher concentration of kaempferol (bound form) was measured in flowers (C. nodosa; 0.38 mg/gdw > C. renigera; 0.20 mg/gdw > C. pumila; 0.14 mg/gdw) and minimum concentration was measured in stem (C. pumila; 0.02 mg/gdw = C. renigera; 0.02 mg/gdw ) but in stem of C. nodosa was not measured (Table-1).

Similarly the concentration of free form of kaempferol-7-O-glucoside was measured in flowers (C. nodosa; 0.26 mg/gdw > C. renigera; 0.25 mg/gdw > C. pumila; 0.16 mg/gdw), and minimum levels was observed in stem (C. nodosa; 0.02 mg/gdw < C. pumila; 0.03 mg/gdw), but in stem of C. renigera, it was not measured. The higher concentration of kaempferol-7-O-glucoside (bound form) was measured in flowers (C. nodosa; 0.16 mg/gdw > C. renigera; 0.10 mg/gdw > C. pumila; 0.05 mg/gdw) but in stem and root of selected species was not measured (Table-1).

CONCLUSION:

The above results indicates that higher levels of both free and bound form of all the isolated flavonoid (kaempferol, quercetin and kaempferol-7-O-glucoside were found in flowers and minimum levels were measured in roots except kaempferol-7-O-glucoside that however its minimum level were observed in stem.

ACKNOWLEDGEMENT:

We are very thankful to Head , Deparment of Botany to provide support and help, we feel indebted to Mr.Suman Kumar for his help in spectrometry and chemical related issues..

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Received on 28.03.2011 Modified on 05.04.2011

Asian J. Research Chem. 4(5): May, 2011; Page 818-821