Pharmacognostical Studies on Leaves of Allamanda cathartica with Detail Physicochemical and Phytochemical Evaluation

 

Kirteebala P. Pawar1*, Milind J. Bhitre2, Priyanka V. Kalamkar1, Mohan K. Kale1

1Department of Pharmaceutics, Assistant Professor, Konkan Gyanpeeth Rahul Dharkar College of Pharmacy and Research Institute, Karjat.

2 Department of Pharmaceutical Chemistry, Professor, C. U. Shah College of Pharmacy, Santacrutz, Mumbai *Corresponding Author E-mail: kirtee.khairnar@rediffmail.com

 

ABSTRACT:

Allamanda cathartica L. is an evergreen vine like woody shrub which is a genus of mostly climbing shrub of Apocyanaceae family. In traditional medicine in moderate doses, the leaves are used as a cathartic, their infusion or extract is given in colic. Therefore in this context the detailed pharmacognostic study of leaves with reference to physicochemical and phytochemical evaluation has been carried out with aim to establish its pharmacognostical standards. The phytochemical studies of methanolic extract of leaves reveals the presence of carbohydrates, reducing sugars, hexose sugar, Saponin glycosides, flavonoids, alkaloids, amino acids, phenolic compounds, steroids, proteins.

 

KEYWORDS: Allamanda cathartica, methanolic extract, leaves, physicochemical evaluation, phytochemical evaluation, HPTLC.

 

 


INTRODUCTION:

The genus Allamanda belongs to family Apocyanaceae comprises of many vatieties like A. blanchetti and A .violacea1. It is an erect, evergreen, variable shrub; up to 4 meter in height, widely grown in Indian gardens.The plants does not tolerate shade, salty or alkaline soils. They are highly sensitive to frost1. Allamanda cathartica L. (Apocyanaceae) is commonly known as Angel’s trumpet, Yellow bell1. It is native to South and Central America and West Indies. It is distributed throughout tropics. It is in many parts of Central, Eastern, and Southern India and in Andaman Islands. Roots, shoots, leaves, flowers, fruits, milky sap and whole plant are used2.

 

Fig. No. 1: Plant of Allamanda cathartica Linn.

 

DESCRIPTION: 1, 3

Leaves:

They are whorled, obviate to oblong lanceolate, 8-12*2.5-4.0cm, wavy and which may be either opposite or whorls of three or four.

 

Flowers:

Flowers are very large, usually yellow, in few flowered terminal and auxiliary cymes. Corrolla with short tubular base, then suddenly companulate, lobes rounded, contorted to the left, throat with a ring of ciliate scales. Overy one celled with two parietal placentae and many ovules.

Fruits:

These are globus with numerous stout, green prickles, finally two valved.

 

Seeds:

Capsules are subglobose, 4 to 6 cm in diameter and densely prickled. They contain many tan, flattened, winged seeds.

 

MATERIALS AND METHODS:

The plant material used in this investigation consists of leaves of Allamanda cathartica Linn (Family: Apocynaceae) which was collected from local market and was authenticated by Nicholas Piramal Pvt. Ltd., Goregaon, Mumbai. Extraction was performed by continuous extraction using Soxhlet extractor. Methanol was the solvent used for extraction.

 

Preliminary physico-chemical evaluation4:

Evaluation of drug means confirmation of its identity and determination of its quality, purity and detection of the nature of adulteration. The evaluation of a crude drug is necessary because of three main reasons: biochemical variation in the drug, deterioration due to treatment and storage and substitution and adulteration. Preliminary Phytochemical screening is a part of chemical evaluation. The qualitative chemical test is useful in detection of adulteration. Air dried leaves were used for quantitative determination of loss on drying, total ash, acid insoluble ash, alcohol soluble extractive values according to standard procedure of Indian Pharmacopoeia (Anonumous, 1996).

 

Phytochemical investigation5:

The plant is biosynthetic laboratory, not only for chemical compounds such as carbohydrates, proteins and lipids, but also for a multitude of compounds like glycosides, alkaloids, volatile oils, tannins etc that exert physiological and therapeutic effects. The compounds that are responsible for medical property of the drug are usually secondary metabolites. A systemic study of a crude drug embraces, through consideration of primary and secondary metabolites. Thus the plant material is subjected to preliminary phytochemical screening for the detection of various plant constituents.24 Phytochemical analysis of extract was conducted following the procedure of Indian Pharmacopoeia (Anonymous, 1996). By this analysis, the presence of several phytochemicals likes alkaloid; flavonoid, tannin, saponin, protein and carbohydrate were tested.

 

High performance thin layer chromatography (HPTLC):

High Performance Thin Layer Chromatography studies are major advancement of TLC principles. HPTLC studies were carried out to obtain the finger print of methanolic extract of Allamanda cathartica Lin. extract, which offers better quantitative resolution and requires shorter time.

 

The conditions followed for HPTLC development were as follows:

Conditions of applications:

Instrument used                      : Linomat IV applicator.

Test plates                    : Silica gel GF254 (E-Merk) precoated aluminium plates.

Format                                       : 10 X 10 cm.

Spotting volume                        : 2µl, 4µl and 10µl.

Band size                                   : 8 mm.

Speed of drying time                 : 15 sec/µl.

Nitrogen pressure                      : 2 psi.

 

Conditions of chromatography:

Solvent system                  : All analytical grade solvents.

Separation technique         : Ascending type.

Development chamber      : Twin trough glass chamber.

Distance of migration        : 9 cm.

 

Conditions of scanning:

Instrument: Camag Scanner IV.

Mode        : Absorption / reflection with deuterium lamp.

Wavelength: UV-254 nm, 366 nm, 580 nm.

Slit dimension: 6X 0.30 mm.

 

HPTLC fingerprints of the fractions were obtained using the above chromatographic conditions.

 

RESULTS AND DISCUSSION:

Preliminary physico-chemical evaluation:4

It is necessary that every medicinal plant prior to its use be identified and standardized for its various physicochemical parameters. Air dried leaves were used for quantitative determination of phytochemical values. The ash values, extractive values, percentage yield, color and consistency of methanolic extract of Allamanda cathartica L. were determined five times as per WHO recommendations. (Table No.1, 2)


 

Table No. 1: Ash and extractive values:

Drug

Total Ash Value

Acid Insoluble Ash

Water Soluble Ash

Sulphated Ash

Water Extractive

Alcohol Extractive

Allamanda cathartica

9.33%w/w

3.70%w/w

5.25%w/w

6.50%w/w

18.20%w/w

4.88%w/w

 

Table No. 2: Percentage yield, color, consistency of extracts of Allamanda cathartica Linn.

Extraction solvent

% Yield

Color

Consistency

Methanol

 32.06 %

Greenish Brown

Viscous

 

 


Phytochemical Investigation5:

The preliminary phytochemical screening of methanol extract showed the presence of carbohydrate, saponin glycoside, coumarin glycoside, alkaloids and proteins.(Table No. 3)

 

Table No. 3: Preliminary phytochemical screening for Allamanda cathartica Linn.

Name of the test

Methanolic extract

Carbohydrates

+

Reducing sugar

+

Monosaccharides

+

Pentose sugar

+

Hexose sugar

+

Non reducing polysaccharides

-

Glycosides

 

Cardiac glycosides

-

Anthraquinone glycosides

-

Saponin glycosides

+

Coumarin glycosides

-

Flavonoids

+

Alkaloids

+

Phenolic compound

+

Steroids

+

Proteins

+

Amino acids

+

 

High performance thin layer chromatography (HPTLC):

TLC plate were examine under ultra violet light at 254 nm, 366 nm, 580 nm. The mobile phases used for methanolic  extract  was as mentioned in Table No.4.The fingerprints obtained for extract were  as shown in Figure No.2.Rf  values and color of the bands obtained were recorded. (Table 5, 6, 7)

 

Table No.4: The mobile phases of methanolic extract and it’s TLC plates for the plant extract Allamanda cathartica Linn.

Sr. no.

Extract

Mobile phase

Spot observed

1

Methanol

Toluene : Chloroform: Ethanol  [8:8:2]

13

 

Image at 366 nm             Image at 254 nm         Image at 580nm

Fig. No.2: Fingerprints of methanolic extract of Allamanda cathartica Linn.


 

 

Table No.5:   HPTLC peak area of methanolic extract (10µl) of Allamanda cathartica Linn. at 254 nm.

Peak

Start Rf

Start Ht

Max Rf

Max Ht

Max %

End Rf

End Ht

Area

Area %

1

0.04

28.7

0.06

159.9

12.19

0.07

2.2

1554.0

3.75

2

0.08

7.6

0.09

23.9

1.82

0.11

0.6

266.5

0.64

3

0.18

6.8

0.21

21.0

1.60

0.23

1.6

432.5

1.04

4

0.29

14.0

0.37

302.5

23.05

0.41

27.9

9431.0

22.73

5

0.41

28.0

0.45

134.6

10.26

0.47

55.9

3741.3

9.02

6

0.47

56.0

0.48

66.4

5.06

0.50

50.3

1179.9

2.84

7

0.50

50.7

0.54

118.7

9.04

0.55

100.1

3235.6

7.80

8

0.55

100.5

0.56

123.2

9.39

0.59

13.8

2364.9

5.70

9

0.59

13.8

0.62

44.2

3.37

0.66

3.2

1256.6

3.03

10

0.70

2.9

0.87

205.5

15.66

0.92

100.3

15504.4

37.37

11

0.92

100.3

0.94

112.2

8.56

0.97

1.7

2523.3

6.08

 

 

Table No.6:  HPTLC peak area of methanolic extract (10µl) of Allamanda cathartica Linn. at 366 nm.

Peak

Start Rf

Start Ht

Max Rf

Max Ht

Max %

End Rf

End Ht

Area

Area %

1

0.09

0.1

0.11

19.5

1.74

0.13

4.6

256.1

0.99

2

0.15

3.8

0.18

56.4

5.05

0.20

22.3

1026.4

3.96

3

0.20

22.3

0.22

41.

3.71

0.24

15.5

747.9

2.89

4

0.24

15.3

0.30

130.6

11.69

0.32

40.2

3347.3

12.93

5

0.32

40.3

0.33

42.8

3.84

0.36

0.9

690.5

2.67

6

0.36

1.0

0.39

315.2

28.22

0.41

162.1

6475.1

25.01

7

0.41

162.2

0.43

283.3

25.37

0.48

54.1

7192.9

27.78

8

0.48

54.9

0.51

73.3

6.56

0.53

1.0

2184.1

8.43

9

0.54

1.3

0.56

38.3

3.43

0.56

36.5

519.8

2.01

10

0.58

38.6

0.60

50.5

4.52

0.64

20.1

1781.9

6.88

11

0.64

19.8

0.68

37.8

3.39

0.71

0.4

1123.7

4.34

12

0.71

0.5

0.74

27.7

2.48

0.77

1.0

547.8

2.12

 

 

Table No.7:  HPTLC peak area of methanolic extract (10µl) of Allamanda cathartica Linn. at 580 nm.

Peak

Start Rf

Start Ht

Max Rf

Max Ht

Max %

End Rf

End Ht

Area

Area %

1

0.04

1.8

0.05

127.1

5.91

0.07

0.6

1312.9

1.61

2

0.07

0.4

0.08

11.5

0.53

0.09

0.4

86.7

0.11

3

0.10

0.0

0.12

34.4

1.60

0.15

0.2

633.0

0.78

4

0.15

0.1

0.18

149.0

6.93

0.20

0.9

2589.2

3.18

5

0.21

0.0

0.25

64.5

3.00

0.29

7.1

1345.3

1.65

6

0.29

7.3

0.36

247.6

11.51

0.39

110.1

9220.3

11.33

7

0.41

112.5

0.43

153.1

7.11

0.45

101.1

3612.4

4.44

8

0.45

101.2

0.47

184.0

8.55

0.49

137.5

5309.4

6.53

9

0.50

138.8

0.56

366.8

17.05

0.56

362.6

12392.4

15.23

10

0.57

363.0

0.58

383.0

17.80

0.62

163.0

12144.5

14.93

11

0.66

78.7

0.69

128.0

5.95

0.74

70.1

5428.3

6.67

12

0.74

69.9

0.86

302.2

14.05

0.97

7.4

27288.6

33.54

 


 

CONCLUSION:

In conclusion, the physicochemical and phytochemical evaluation and HPTLC fingerprinting of leaves of Allamanda cathartica L. can assist as a relevant source of information for further medicinal studies of this plant in future exploration.

 

REFERENCES:

1.       Wealth of India – a dictionary of Indian Raw Material and Industrial Products, National Institute of Science, CSIR, New Delhi Publication, Volume 1, 1998; 164-165.

2.       Rastogi, Malhotra, Compendium of Indian Medicinal Plants, Drug Research Perspective: A, CDRI series 1980, volume 3; 30.

3.       A.K. Nadkarni’s Indian Materia Medica, Popular Prakashan, volume 1; 62.

4.       Arya Vaidya Sala, Kottakkal: Indian Medicinal Plants – a compendium of 500 species, Orient Lonfman Ltd Publication, Volume 4; 290-296.

5.       The Ayurvedic Pharmacopoeia of India, Govt. of India Ministry of Health and Family, New Delhi, 1st edition, volume 1, 2001; 234-255.

6.       C. K. Kokate, A. P. Purohit and S. B. Gokhale: Pharmacognosy, 5th edition, Nirali Prakashan, Pune, 1997; 106-108.

7.       R. D. Chaudhari: Herbal Drugs Industry, 1st edition, 1996; 1-3.

 

 

 

Received on 07.04.2015       Modified on 19.04.2015

Accepted on 26.04.2015      ©A&V Publications All right reserved

Res.  J. Pharmacognosy & Phytochem. 7(2): April-June 2015; Page 69-72

DOI: 10.5958/0975-4385.2015.00013.8