Phytochemical Analysis of Methanolic Extract of Roots of Kalanchoe pinnata by HPLC and GCMS

 

Quazi Majaz*, Sayyed Nazim, Shaikh Siraj, Mohd. Zameeruddin and Rahil Khan.

Ali Allana College of Pharmacy, Akkalkuwa, Dist- Nandurbar [MS].

*Corresponding Author E-mail: quazimajaz@gmail.com

 

 

 


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, antiinflammatory9, 11and 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), 5m 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), 5m 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 180C, 180-260C at 3C/min, 5 min in 260C, 260-

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

temperature, 280C detector temperature, 290C.

Volume injected : 1 L of sample

Ionization potential : 70 eV

Ion source temperature: 290C.

 

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[mV*s]

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[mV*s]

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

 

 


4. CONCLUSION:

Preliminary phytochemical screening of methanolic extract of K. Pinnata shows the presence of Steroids, Saponins, Alkaloids, Glycosides, Flavonoids and Tannins. HPLC analysis shows the presence of quercetin, myrcetin, cholesterol and beta sitosterol. While but GCMS data show only steroids and sugar moieties, no molecule from flavonoids. The possible reason should be due lack of derivetisation technique which degrade the flavonids on heating into glycon and aglycon moiety.

 

From these above phytochemical investigation it was concluded that chloform extract of Kalanchoe pinnata root contain various flavonoids and steroids. Which will leads further pharmacological investigation of this plant.

 

 


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

Accepted on 23 01.2011 AJRC All right reserved

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