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 investigated^[1]. Cassia nodosa 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^[2,3]. Plants used as source of indigenous medicines due presence of various secondary metabolites. In the present investigation, new flavonoids were isolated and their antifertility activities were carried out. Among natural phenolics, the flavonoids forms the largest group and more than 2,000 flavonoids are reported among woody and non-woody plants^[4-7].

A number of plants have been screened for the presence of flavonoids by many researchers^[8-11]. Thus traditional medicinal plants derived antioxidants may protect against a number of diseases and reduce oxidation processes in food systems^[12,13]. In order to establish this, it is imperative to measure the markers of baseline oxidative stress particularly in human health, disease and examine how they are affected by supplementation with pure compounds or complex plant extracts from the traditional medicinal plants^[14]. The data so far generated clearly sets the basis for a clear understanding of the phytochemistry of the plant with 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^[13,15]. The flavonoids have utmost importance in providing strength and resistance to the plant against various diseases but also exhibit various biological and pharmacological activities^[16-19]. A survey of literature suggested that the flavonoids are found both, in free and bound forms, the former being in higher levels than the later^[20].

In the present study a new flavonoid kampferol-7-O-glucoside with the other flavonoids were isolated from C. nodosa and characterized them by spectral studies and chromatography. Antifertility activity of these isolated compounds was carried out by the established protocols. Plants have served as natural sources of anti-fertility substances. Several Cassia species have been reported to be used in the aid of family planning in ethnomedicine^[21]. However, there is no systemic study on the anti-fertility activity of the selected Cassia species reported so far. Therefore, in the present study, an attempt has been made to screen the anti-fertility activity of the selected Cassia species, on account of their repetition in ethnomedicine.

MATERIAL AND METHODS:

Collection and Identification

C. nodosa is an ornamental tree which is found wildly usually in the gardens and road sides. Plant species collected from central park of Jaipur (Rajasthan), India. The plant was identified at Herbarium, Department of Botany, University of Rajasthan, Jaipur and their voucher specimen (No. 3024) has been deposited in the Herbarium.

Processing and Extraction

The plant parts (root, stem, leaves, flowers and pods) of C. nodosa were studied for the determination of flavonoids. Plant sample was extracted with 80% methanol^[22] and the concentrated extract was fractionated with pet. ether (Fraction I). diethyl ether (Fraction II) and ethyl acetate (Fraction III), respectively. Fr. I was rejected in each case because of being rich in fatty components whereas, Fr. II and Fr. III were analyzed 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^[9]. Thin layer chromatography of Fr. II and Fr.

IV were done along with the standard markers^[7]. 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^[23]. Fluorescent spots of the samples coinciding with the standard. Bands are matched with the standard at kaempferol (R_f0.85), 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^[24]. Using spectrophotometric methods of the quantification was made of kaempferol, quercetin and kaempferol-7-O-glucoside, respectively^[6,25].

Antifertility Test

Animal Treatment

Wistar rats, weighing 150-160 g obtaining from Jamia Hamdard University, New Delhi were used. Animals were housed in steel cages and maintained under standard condition (12 hrs. light/ 12hrs. dark cycle; 25±3°C; 35-60% relative humidity). Rat feed (Hindustan Lever Ltd.) and tap water was provided ad libitum.

Experimental Design

Male rats of proven fertility were divided into two groups of 10 each. One group was treated with C. nodosa fraction (50 mg/ kg/ b.wt./ day) for 60 days. The control group existed of rats receiving vehicle (distilled water 0.5 ml/ day) for 60 days. On day 60 testes, epididymides, seminal vesicle, ventral prostate, liver and adrenal glands were removed cleared off fat and connective tissue and weighed.

Fertility Test

Female rats 10-12 weeks old weighing between 150-160 g were used for the investigation. The fertility of each male was assessed by natural mating with three proestrus and virgin females before, during and after the treatment. The presence of spermatozoa in vaginal swab of the cohabited females was used as evidence of mating. The females were separated. The implantation sites was recorded on day 16 after mating through laprotomy and fertility was calculated in control as well as treated group.

Statistical calculation

All the values of body/ organs weights, biochemical estimation histometry and testicular dynamics were expressed in terms of mean ≡ standard error. The treated groups were compared to controls using the student's 't' test^[26].

RESULT AND DISCUSSION:

Structure Elucidation of Isolated Flavonoids

In the present investigation, flavonoids profile has been studied in vivo of C. nodosa, where kaempferol-7-O-glucoside, kaempferol and quercetin from different plant parts of C. nodosa 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 suitable solvents and evaporated yielding three flavonoids kaempferol-7-O-glucoside, kaempferol and quercetin^[19]. The spectral analyses of the active constituent, kaempferol-7-Oglucoside; kaempferol and quercetin from the different plant parts of selected C. renigera are shown below: -

Kaempferol-7-O-glucoside:

Brownish needles on crystallization (m.p. 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;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);

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).

Fig. 1: Structure of Kaempferol-7-O-glucoside

Kaempferol:

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), 1700 (C=O), 1600, 1610, 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)

Fig. 2: Structure of Kaempferol

Quercetin:

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), 1680 (C=O), 1610, 1610, 1560, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010;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); 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. 3: Structure of Quercetin

Concentration 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 flavonoids was observed in the flowers (free = 0.98 mg/gdw, bound = 0.41 mg/gdw). The minimum concentration of both free and bound flavonoids was observed in roots i.e. 0.19 mg/gdw and 0.10 mg/gdw, respectively. Among the isolated flavonoids, kaempferol was recorded in more concentration in flower (0.50 mg/gdw, free form) than the isolated other flavonoids. In the bound form kaempferol was recorded in higher concentration (0.18 mg/gdw) in flowers also. Over all in the whole plant the isolated free flavonoids were recorded in higher concentration (2.04 mg/gdw) than the bound flavonoids (0.79 mg/gdw).

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

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 nodosa.

Plant species	Plant parts	Free (F)				Bound (B)				Total (F+B)
Plant species	Plant parts	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total	Quercetin	Kaempferol	Kaempferol-7-O-glucoside	Total
*C. nodosa*	Root	0.04	0.11	0.06	0.23	0.04	0.04	0.04	0.14	0.08	0.15	0.10	0.37
	Stem	0.08	0.12	0.07	0.27	0.08	0.06	0.04	0.18	0.16	0.18	0.11	0.45
	Leaves	0.14	0.29	0.08	0.51	0.13	0.10	0.06	0.27	0.27	0.37	0.14	0.78
	Flowers	0.28	0.49	0.20	0.98	0.14	0.18	0.09	0.41	0.42	0.68	0.29	1.39
	Pods	0.17	0.20	0.08	0.45	0.10	0.07	0.04	0.21	0.27	0.27	0.12	0.66
	Total	0.73	1.22	0.49	2.04	0.51	0.43	0.27	0.79	1.24	1.65	0.76	3.65

Table 3 : Effect of Kaempferole-7-O-glucoside on body weights and organ weights.

Treatment	Body weight(g)	Organ weight (mg/100 g b.wt.)
Treatment	Body weight(g)	Testis	Epididymides	Seminal vesicle	Ventral postrate
Control	223.84 ± 5.85	1318.88 ± 7.86	489.63 ± 3.26	670.03 ± 13.52	368.86 ± 9.51
Kaempferole-7-O-glucoside	229.90 ± 16.89ns	989. 98 ± 13.85	405.34 ± 5. 19	579.90 ± 8. 37	296. o6 ± 4.52

Values are mean ± SEM(n=10); ns= non significant; p<0.001vs control.

Table 4 : Effect of Kaempferole-7-O-glucoside on sperm motility, density and fertility in rats

Treatment	Sperm motility % (Cauda epididymides)	Sperm Density (million/ml)		Fertility %
Treatment	Sperm motility % (Cauda epididymides)	Testis	Cauda epididymides	Fertility %
Control	69.23±5.46	6.08 ± 10.92	68.54 ± 16.85	100 (+) ve
Kaempferole-7-O-glucoside	18.23±1.05**	1.45±9.95**	6.65±10.03	95 (-) ve

Values are mean ± SEM (n = 10); ** P < 0.001 vs Control; Standard error mean

Antifertility activity

Plants have served as natural sources of anti-fertility substances. The genus Cassia, have been reported to be used in the aid of family planning in ethno medicine^[27]. However, no systemic study on anti-fertility activity of the selected Cassia species was reported so far. Therefore, in the present study, an attempt has been made to screen the anti-fertility activity of the selected medicinal plants on account of their repetition in ethno medicine. Antifertility effect of various flavonoids of Cassia species has been reported.

The survey of literature revealed that no attention has been paid to activity of kaempferol-7-O-glucoside isolate from C. nodosa on the reproductive system of male rats. In the present investigation we are reporting the antifertility activity of the kaempferol-7-O-glucoside C. nodosa on male rats.

The oral administration of kaempferol-7-O-glucoside at the dose level 50mg/ kg/ b.wt/ day for 60 days did not cause any significant change in the body weight of treated rat but the weight of testes (P<0.001), epididymides (P<0.001), seminal vesicle (P<0.001) and ventral prostrate(P<0.001) reduced in significant manner (Table 3).

Kaempferol-7-O-glucoside feeding to rats significantly reduced sperm concentration of testes and epididymisdes (P<0.001). The motility of the cauda epididymal sperm was also reduced significantly (P<0.001). The extract reduced the fertility of male rats by 95% (Table 4).

The results obtained in Table 3 and 4 provides clear evidence that the plants showed pregnancy interruption on rats was dose dependent and proved to be more effective only at early stages of pregnancy as supported by the experimental observations^[28]. In general kaempferol-7-O-glucoside are known to produce anti-fertility activity^[29]. The selected plants revealed the presence of flavonoids. Perhaps, anti fertility activity of selected plants depends on the length of the pregnancy and quantity of the active principles present in the flavonoids. These results thus, validate the use of the plants as anti fertility agents used in ethno medicine. However, further experiments including estrogenic evaluation are required to elucidate its mechanism of action.

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 flavonoids 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^[30-32].

On the basis of above results, it is concluded that the C. nodosa 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.

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Received on 15.06.2012 Modified on 19.07.2012

Asian J. Research Chem. 5(8): August, 2012; Page 985-989

Colours by chromatogenic sprays colour