1. INTRODUCTION:

Various species of KALANCHOE are used medicinally in Indo-China and Philippines Islands, whereas Kalanchoe pinnata Pers. (Family Crassulaceae) is naturalized throughout the hot and moist parts of India. The leaves and bark is bitter tonic, astringent to the bowels, analgesic, carminative, useful in diarrhoea and vomiting ⁵. Antiulcer¹⁰, antiinflammatory^{9, 11}and antimicrobial activity¹ of leaf extract was reported. Oral treatment with leaf extract significantly delayed onset of disease in BALB/c mice infected with Leishmania amazonensis as compared to untreated mice or mice receiving K. pinnata by the intravenous or topical routes ². Potent cytotoxic compounds bersaldegenin-1,3,5-orthoacetate¹⁶ and bufadienolide-bryophyllin B ¹⁵ were isolated. Other chemical constituents from this plant are bryophyllol, bryophollone, bryophollenone, bryophynol and two homologous phenanthrene derivatives 2(9-decenyl)-phenanthrene (I) and 2-(undecenyl)-phenanthrene (II) from leaves¹². Isolation and structure elucidation of 24-epiclerosterol [24(R)-stigmasta-5, 25-dien-3β-ol], 24(R)-5α-stigmasta-7, 25-dien-3β-ol, 5α-stigmast-24-en-3β-ol and 25-methyl-5α-ergost-24 (28)-en-3β-ol from aerial parts was done¹³. This species is also included in the plants species, which are used by the tribals of Kerala for treating cancer symptoms⁶ Juice of the fresh leaves is used very effectively for the treatment of jaundice in folk medicines of Bundelkhand region of India, but no systemic study to assess this activity has been carried out.

As the aerial parts of plant have many pharmacological activity but roots of this plant was not focused yet hence the present investigations were carried out to evaluate phytoconstituent of roots of plant with the help of HPLC and GC-MS, which will help for further pharmacological evaluation.

2. MATERIALS AND METHODS:

2.1. Collection of plant material:

The roots of Kalanchoe pinnata was collected from Satpuda hills near Akkalkuwa, Dist: Nandurbar, Maharashtra, India, in June 2010, cleaned and dried at room temperature in shade and away from direct sunlight. The plant authenticated by T. Chakraborthy, Deputy Director Botanical Survey of India, Koregaon Road Pune, by comparing morphological features and a sample voucher specimen of plant was deposited for future reference (Voucher specimen number QMAKP1).

2.2. Preparation of extract:

The root of Kalanchoe pinnata was collected and dried in the shade and then pulverized in a grinder. The powdered drug was utilized for extraction. Material was passed through 120 meshes to remove fine powders and coarse powder was used for extraction. A method described in Mukherjee was used for extraction of powdered plant. Extraction was done by methanol.⁷

2.3. Preliminary phytochemical screening:

The extract was then subjected to preliminary phytochemical screening to detect the presence of various phytoconstituent. The results show presence of Steroids, Saponins, Alkaloids, Glycosides, Flavonoids, Tannins and carbohydrates in the methanolic extract.⁴

2.4. HPLC analysis for flavonoids:⁸

Column: Hypersil- ODS Column (250mm × 4.6mm), 5µm particle size

Mobile phase: Acetonitrile: Phosphate buffer pH 2.4 (25:75)

Flow rate: 1.2 ml/ min

Detection: 266 nm

2.5. HPLC analysis for steroids:¹⁴

Column: Hypersil- ODS Column (250mm × 4.6mm), 5µm particle size

Mobile phase: Methanol: Water (95:5)

Flow rate: 1ml/ min

Detection: 210 nm

2.6. GC-MS Analysis:³

The GC-MS analyses were carried out in Perkin Elmer, Auto system XL GC+.

Carrier gas : helium with a flow rate of 0.7 mL/min;

Column temperature: 5 min in 180°C, 180-260°C at 3°C/min, 5 min in 260°C, 260-

280°C at 0.2°C/min, and finally 5 min in 280°C; injector

temperature, 280°C detector temperature, 290°C.

Volume injected : 1 µL of sample

Ionization potential : 70 eV

Ion source temperature: 290°C.

3. RESULTS AND DISCUSSION:

3.1. HPLC analysis for flavonoids:

Analysis of flavonoids in chloroform extract of plant shows 17 compounds (Table 1 and Graph 1) after comparing with standard retention time of various flavonoids, it shows peak at 7.2500 and 14.5667 which is nearer to myrcetin and quercetin respectively.⁸

Graph 1 Chromatogram of HPLC for methanolic extract for flavonoids

3.2. HPLC analysis for steroids:

Analysis of flavonoids in chloroform extract of plant shows 9 compounds (Table 2 and Graph 2) after comparing with standard retention time of various flavonoids, it shows peak at 7.5890 and 9.7583 which is nearer to cholesterol and beta sitosterol respectively.¹⁴

Graph 2. Chromatogram of HPLC for methanolic extract for steroids

3.3. GC-MS Analysis

After preliminary phytochemical investigation and HPLC analysis shows the presence of various phytoconstituent in methanolic extract and investigation done by GCMS.

Gaschromatograph of methanolic extract shows the presence of 17 phytoconstituent (Table 3 and Graph 3) which are subjected to mass spectroscopy. The mass spectroscopy of these peaks does not give any prominence result it shows only presence of steroids and various sugar molecules, here ms data of methanolic extract for peak at 37.039 RT (Table 4 and Graph 4). The list of possible phytoconstituent is given by checking compound in Nist and Nbs library.

Graph 3 GC chromatogram of methanolic extract

Table 1. Results of HPLC for methanolic extract for flavonoids

No.	Name	RT[min]	*Area[mVs]**	Area%	TP	TF	Resolution
1		2.5000	359.7884	45.36	2.5	0.7143	0.0000
2		2.5833	204.2780	25.75	12.8	1.2500	0.0153
3		3.0667	27.0487	3.41	1551.6	0.7222	0.2566
4		3.2000	3.5687	0.45	41.7	1.1250	0.0988
5		3.3667	5.6438	0.71	4617.7	0.9000	0.1299
6		3.5333	19.9530	2.52	92.2	0.8000	1.1111
7		3.6500	14.3980	1.82	0.9	0.7500	1.0000
8		3.9000	33.5674	4.23	4.4	0.8333	0.0183
9		4.7167	10.8464	1.37	3084.1	0.7500	0.1782
10		5.0667	23.4179	2.95	3032.3	1.0500	0.8400
11		5.5667	21.6275	2.73	70.0	0.8750	0.2804
12		6.7833	15.5456	1.96	4082.5	1.0417	0.6697
13		7.2500	13.2003	1.66	4663.5	1.1364	0.9333
14		11.3167	14.5783	1.84	3781.9	1.0800	5.9512
15		14.5667	6.4741	0.82	13073.6	1.0769	4.4318
16		15.4500	12.5182	1.58	4958.5	0.5645	1.0816
17		16.3333	6.7806	0.85	13314.0	1.4231	1.0392
Sum			793.2349

Table 2. Results of HPLC for methanolic extract for steroids

No.	Name	RT[min]	*Area[mVs]**	Area%	TP	TF	Resolution
1		2.2667	958.6914	39.95	64.2	0.5732	0.0000
2		2.9167	532.9800	22.21	138.6	1.0625	0.4065
3		4.5333	151.8046	6.32	256.4	1.0000	1.2933
5		6.2667	135.6579	5.65	6479.0	1.3182	0.7455
6		7.5890	194.7569	8.11	29.7	0.9091	0.4780
7		8.0752	099.5738	4.14	70.0	0.8750	0.2804
8		9.7583	217.3957	9.05	321.4	3.2000	0.5063
9		12.3584	108.7375	4.53	393.9	3.8750	0.3067
Sum			2399.5978

Table 3.Area percent report of methanolic extract

No.	Name	RT	Area (A)	Height	BL	Conc.	Units	A/conc.	m/z	Area%
1		6.989	129,919.7	241,651	MM	0.00		000	TIC	12.62
2		9.995	11,846.3	65,187	MM	0.00		000	TIC	1.15
3		15.826	30,780.6	150,509	MM	0.00		000	TIC	2.99
4		16.871	46,183.8	243,724	MM	0.00		000	TIC	4.49
5		17.109	44,046.6	280,523	dd	0.00		000	TIC	4.28
6		17.274	95,515.7	396,261	MM	0.00		000	TIC	9.28
7		20.006	8,754.9	68,441	MM	0.00		000	TIC	0.85
8		20.428	32,470.6	247,982	bb	0.00		000	TIC	3.15
9		20.629	19,745.3	155,843	bb	0.00		000	TIC	1.92
10		22.151	19,471.7	160,087	MM	0.00		000	TIC	1.89
11		22.353	49,589.5	336,108	dd	0.00		000	TIC	4.82
12		22.536	7,403.2	60,260	MM	0.00		000	TIC	0.72
13		31.648	19,935.0	134,996	db	0.00		000	TIC	1.94
14		33.042	50,307.4	205,392	MM	0.00		000	TIC	4.89
15		33.518	153,983.9	389,866	dd	0.00		000	TIC	14.96
16		33.940	284,270.3	702,377	MM	0.00		000	TIC	27.61
17		37.039	25,346.9	141,342	MM	0.00		000	TIC	2.46

Table 4. List of probable compound of methanolic extract for peak at 37.039 RT

Hit	Rev	For	Compound name	M.W	Formula	CAS	Library
1	842	690	Beta-sitosterol acetate	456	C₃₁H₅₂O₂	915-05-9	Nbs
2	830	738	Gamma-sitosterol	414	C₂₉H₅₀O	83-47-6	Nbs
3	807	567	Campesterol	400	C₂₈H₄₈O	474-62-4	Nbs
4	803	618	Cholesterol	386	C₂₇H₄₆O	57-88-5	Nbs
5	786	624	Dihydrotachysterol	398	C₂₈H₄₆O	67-96-9	Nbs
6	785	597	Methyl(25RS)-3beta acetoxy-5-cholestin-26-oate	472	C₃₀H₄₈O₄	103160-13-0	Nist
7	777	678	Cholesta-3,5-diene	368	C₂₇H₄₄	747-90-0	Nbs
8	775	703	Cholesta-3,5-diene	368	C₂₇H₄₄	747-90-0	Nbs
9	773	549	Cholesterol	386	C₂₇H₄₆O	57-88-5	Nbs
10	772	674	Cholesta-3,5-diene	386	C₂₇H₄₄	747-90-0	Nbs
11	756	528	Cholesta-5,22-dien-3-ol, (3 beta)-	368	C₂₇H₄₄O	34347-28-9	Nist
12	736	532	Cholesta-5- ene, 3methoxy-, (3 beta)-	384	C₄₇H₈₂O₂	1174-92-1	Nbs
13	727	483	Stigmast-5-en-3ol,oleate	400	C₂₈H₄₈O	1000154-66-5	Nist
14	721	608	Beta-sitosterol acetate	678	C₃₁H₅₂O₂	915-05-9	Nist
15	716	574	21-acetoxypregnalone	456	C₂₃H₃₄O₄	566-78-9	Nist
16	715	628	Stigmastan-3,5-diene	374	C₂₉H₄₈	1000214-16-4	Nist
17	709	549	Cholesta-5- ene, 3-ol, (3 beta)-nonanoate	396	C₃₆H₆₂O₂	1182-66-7	Nist
18	709	452	Cholane-5,20(22)-diene-3b-phenoxy	526	C₃₀H₄₂O	10014-88-6	Nist
19	707	490	Stigmasterol	418	C₂₉H₄₈O	83-48-7	Nbs
20	701	436	Stigmastan-3-ol,5-chloroacetate	412	C₃₁H₅₃O₂Cl	55724-19-1	Nist

Graph 4 MS data for methanolic extract for peak at 37.039 RT

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Received on 21.12.2010 Modified on 13.01.2011

Asian J. Research Chem. 4(4): April, 2011; Page 655-658706