Pharmacognostical Evaluation of Plumeria alba Linn

 

R Radha1*, T Sivakumar1 and S Arokiyaraj2

1 Department of Pharmaceutical Sciences, Madras Medical College, Chennai – 3,

 2Department of Biomedical Engineering, Sathyabama University, Chennai – 119

*Corresponding Author E-mail:  rradha1970@yahoo.co.in

 

ABSTRACT:

Plumeria alba Linn (Apocynaceae) is used in the treatment of ulcers, herpes, scabies and seeds possess haemostatic properties. The bark is bruised as plaster over hard tumours. Only a very limited research has been carried out on the plant, under the present study assumes singular significance and it is supposed to contribute a great deal to the existing literature. The present paper highlights the macroscopic and microscopic characters of leaf, petiole, physico chemical evaluation and preliminary phytochemical studies of the plant (leaves). These observations would be of immense value in the botanical identification and standardization of the drug in crude form. This study would help distinguish the drug from its other species.

 

KEY WORDS:  Apocynaceae Pharmacognosy, HPTLC, Plumeria alba

 


 

INTRODUCTION:

Plumeria alba Linn (Apocynaceae) commonly called White Champa, a small laticiferous tree or shrub, native of tropical America. It is 4.5m high, occasionally grown in the gardens. The plant is mainly grown for its ornamental and fragrant flowers. Leaves lanceolate to oblanceolate, flowers white, fragrant, in corymbose fascicles1. The fruit is edible, latex is applied to ulcers, herpes and scabies and seeds possess haemostatic properties. Moreover its bark is bruised and applied as plaster over hard tumours2, 3.  Whereas the latter taxon finds use as purgative, cardiotonic, diuretic and hypotensive4, 5. Methanolic extract showed antimicrobial activity against Bacillus anthracis, Pseudomonas aeruginosa6. The plant is reported to contain amyrinacetate, mixture of amyrins, ί-sitosterol, scopotetin, the iriddoids isoplumericin, plumieride, plumieride coumerate and plumieride coumerate glucoside7, 8. Bioactive richness of these active constituents /were present in the plant.

 

However Pharmacognosy information about this plant has not been published, particularly the necessary to define quality control procedures of the P. alba as raw material. Hence the present investigation deals the Pharmacognostical evaluation of the Plumeria alba. The study includes morphological and anatomical,

 

determination of physico-chemical constants and the preliminary phytochemical evaluation of the different extracts of Plumeria alba

 

MATERIALS AND METHODS:

Plant Material:

The fresh plant material of Plumeria alba was collected from Puducherry, Tamil Nadu, India during the month of August 2007. The botanical identity of the plant was confirmed at the Botanical Survey India (BSI), Coimbatore, Tamilnadu. A voucher specimen (P157) has been deposited at the Museum of the Department of Pharmacognosy, Madras Medical College, Chennai - 03.

 

Chemicals and Instruments: 

Compound microscope, Camera lucida, stage and eyepiece micrometer, glass slides, cover slips, watch glass and other common glassware were the basic apparatus and instruments used for the study. Photomicrographs in different magnifications of all necessary cells and tissues were taken with Nikon Lab Phot - 2 microscopic Unit. Some crystals, starch grains and lignified wall photographs were taken under polarized light microscope. Solvents viz. Hexane, chloroform, ethylacetate, methanol and reagents viz. phloroglucinol, glycerin, Hcl, chloral hydrate and sodium hydroxide were procured from Ranbaxy Fine Chemicals Ltd., Mumbai, India.

 

Macroscopic and Microscopic Analysis: 

The macroscopy and microscopy of the different parts like leaf, midrib, petiole and stem were studied according to the method of Brain and Turner9. For the microscopical studies, cross sections were prepared and stained as per the procedure of Johansen10. Quantitative microscopy was done as per the procedure given by standard procedures 11-13.

 

Figure 1a

 

Physico – Chemical Analysis:

All parameters are applied on only leaves Physico – chemical analysis i.e. percentage of ash values and extractive values were performed according to the official methods prescribed (Indian pharmacopoeia) 14 and the WHO guidelines on quality control methods for medicinal plant materials (WHO/QCMMPM guidelines) 15. Fluorescence analysis was carried out according to the method of Chase and Pratt16.

 

Figure 2a

 

T. S of midrib enlarged [Ads – Adaxial side, Col -  Collenchyma; Cph – Central phloem; Oph – outer phloem; Pa- Parenchyma cells; VS- Vascular strand; X – Xylem.)

 

Preliminary Phytochemical Screening:

Preliminary phytochemical screening was carried out by using standard procedures 17, 18.

 

HPTLC Profile:

Preparation of extract 1 gm of powdered plant material was extracted in soxhlet apparatus with n-hexane, chloroform, ethyl acetate and methanol, individually on a water bath, filtered and made upto 10 ml in a standard flask.  Samples were applied in a concentration of 20ml using CAMAG LINOMAT IV sample applicator on aluminium sheets precoated with silica gel Merck 60F 254, 0.2mm layer thickness (10 cm x 2 cm). TLC plates which were used as a stationary phase. The extracts are spotted with 3mm band at 5sec/ml using HPTLC automatic injector.

 

Figure 2b, 2c

2

 
 


1. T. S of lamina through largerand the smaller veins

2. T. S of lamina showing the abaxial trichomes

[AbE – Abaxial Epidermis; AdE – Adaxial Epidermis; BSE – Bundle sheath extension; LV –lateral vein; MV – Major vein; Ph- Phloem; PM – Palisade mesophyl; SM – spongy mesophyll; SV – Smaller vein; Tr – Trichomes X- Xylem.]

 

 

The plates were eluted with 20 ml of mobile phase. Mobile phase for n-hexane, chloroform, ethyl acetate and methanolic extract, chromatogram was developed in the ratio of   3: 7 hexane in ethyl acetate. The plates were then scanned densitometrically at various wavelengths. Rf values, peak area and spectrum of each peak were determined for these extracts.

 

RESULTS AND DISCUSSION:

Macroscopic Characters (Fig.1a)

Lateral vein size – 32 -35 pairs; Leaves – Length (22 cm), Width (7.5 cm), Obavate – elliptic Fruits -double follicle, green, woody cylindrical and length.

 

Figure 2d, 2e

 

T.S of the lateral vein with lamina

1.        T.S of the leaf through the leaf margin.

[AbE – Abaxial Epidermis; AdE – Adaxial Epidermis; AdS – Adaxial Side Cu – Cuticle LM – Leaf margin; LV –lateral vein; Ph- Phloem; PM – Phloem mesophyl; PM- Palisade mesophyl; SM – spongy mesophyll; Tr – Trichomes; X- Xylem.]

 

Figure 2f

 

1.      a few paracytic stomata enlarged

[Ec – Epidermal Cell; Gc- Guard cell; Sc – Subsidary cell]

 

Microscopic Characters:

Leaf:

The leaf has very thick midrib and thin lamina arising from the adaxial – lateral portion of the midrib.  The midrib is 1.8mm vertically and 2.5mm horizontally.  The ground tissue is differentiated into outer zone of smaller collenchyma cells and remaining portion being parenchymatous. The collenchyma zone is 150μm in the upper part and 50 ΅m wide in the lower part. Narrow thick walled, circular or lobed laticifers are seen randomly dispersed in the ground tissue.  The vascular system consists of a main, wide, bowl shaped thin strand and two small, less prominent accessory adaxial lateral strands.  The main bowl shaped strand is 1.5mm wide, 150μm thick (fig 2a).  It consists of short, radial fibres of 3-5 angular xylem elements and a thin layer of phloem along the outer metaxylem side.  Within the concavity of the vascular arcs these are numerous small nests of phloem elements which are known as inner phloem.  The xylem elements are 30μm wide.  The laticifers are 70μmwide.

 

Figure 2 g

 

 

 

 

 

1.      T.S of petiole

[AdS – Adaxial Side; EP – Epidermis; GT – ground tissue; IGT- Inner ground tissue; IPH -  inner phloem; Lf – Laticifer; OGT - outer ground tissue; VS – Vascular strand]

 

 

Table 1: Physico Chemical Constants

Parameters

Values % (w/w)

Total ash

14.92

Acid insoluble ash

1.64

Water Soluble ash

8.24

Supphated Ash

11.84

Moisture content

11.41

Loss on drying

7.58

Extractive Values

 

Water soluble extractive

7.2

Alcohol soluble

1.87

 

Lamina:

The lamina is trichomatous on the abaxial side and smooth and glabrous on the adaxial side.  The major lateral veins project prominently into conical abaxial part.  The lateral veins and veinlets have a small cluster of xylem elements and a thin arc of phloem.  The vascular bundles are subtended by thick mass of parenchyma cells which extend into adaxial column up to the upper epidermis (fig 2b, 2c). The adaxial epidermis of the leaf has large cells with thick cuticle.  The cells are mostly squarish or rectangular.  The abaxial epidermis is thin and the cells are narrow and cylindrical.  They are 150μm in height.  The spongy mesophyll consists of several lobed parenchyma cells which are interlinked with each other around the air chambers.

 

Figure 3a, 3b

Leaf powder, Stem powder [Fi- fibre, NLf- narrow laticifer, VE- vessel element, WLf-wide laticifer]

[CI-cell inclusions, Lf- Laticifer]

 

Epidermal Trichomes:

The trichomes are non-glandular or covering type and arise from a group of dilated epidermal cells. The trichomes are multicellular, uniseriate and unbranched.  They are narrow and thick walled with smooth surface (fig 2c). 

 

Leaf margin:

The marginal part becomes slightly thin with rounded bent down edge.  The epidermal layer of the leaf margin is thin, the cuticle is very thick and smooth.  The palisade – spongy parenchyma differentiation is absent in the marginal portion.  It consists of 4 or 5 layers of small, thick walled compact parenchyma cells. The marginal part of the lamina is 150μm thick (2e).

 

Epidermal cells and Stomata:

The epidermal cells are rectangular to polyhedral in shape.  The cells have straight or slightly wavy anticlinal walls. The walls are moderately thick. The stomata are paracytic type; there are two subsidiary cells, lying on the lateral sides of the guard cells. The subsidiary cells may be equal or slightly unequal in size. The guard cells are elliptical with wide stomatal aperture. The guard cells are 40 X 70 ΅m in size (Fig 2f). 

 

 

Table 2: Fluorescence analysis of drug powder

Treatment

Day light

UV (254nm)

UV 365nm

Powder as such

Greenish

brown

Green

Green

Powder + NaoH

 (Aquous)

Reddish

brown

Yellow

Brown

Powder + IN NaoH

 (Alcoholic)

Yellow

Green

Green

Powder + IN Hcl

Paleyellow

Dark brown

Dark brown

Powder + IN HNo3

Dark red

Reddish

green

Reddish green

Powder + Ammonia

Dark

green

Fluorescent

green

Dark green

Powder +Iodine

Dark

green

Fluorescent

green

Light green

Powder + Fecl3

Brown

Dark brown

Dark  brown

Powder + actic acid

Greenish

 brown

Dark brown

Light brown

 

Figure: 4

 

Petiole:

The cross sectional outline of the petiole differs from the basal (proximal) part to the terminal (distal) part. The basal part of petiole is circular with a slight concavity on the abaxial side. It measures 3.1mm horizontally and 2.6mm vertically. The ground tissue occupies the major portion of the petiole. It consists of outer wider zone of small, compact, fairly thick-walled cells and remaining regions have larger, circular, less compact parenchyma cells. The vascular tissue form a wide bowl shaped out-line which is 1.5mm wide and 60΅m thick.  Phloem is in the continuous line. Laticifers are distributed throughout the ground tissue; they are more abundant in central portion, inside the vascular strand. Apart from the outer phloem, there are numerous scatted irregular masses of phloem strands distributed along the inner boundary of the xylem arc. Distal part of the petiole has two thick cylindrical adaxial wings and wide, shallow adaxial semicircular round (fig 2g).

 

Figure: 5

 

Table – 3: Quantitative Microscopic Data

Parameters

Values

Stomal number

11.7 sq.mm

Stomatal Index

21.70%

Veinislet Number

15.4 / sq.mm

Veinlet Termination Number

29.4 / sq.mm

 

Powder microscopic observations:

The leaf powder is dark green in colour with an unpleasant order and bitter in taste. On microscopically examination the powder showed numerous unicellular covering trichomes are seen. On maceration the stem powder shows the following elements.

 

Stem fibres:

The xylem fibres are libriform type. They have thick lignified walls and wide lumen. The central portion of the fibre is wider and two ends of the fibre is tapering. Lateral wall pits are not evident. The fibres are 700-750΅m long and 20΅m wide in the middle portion.

 

Laticifers:

Laticifers are quite abundant in the stem and leaf powders. The wide laticifers are 80mm in diameter, the narrow laticifer are less than 10 mm wide (Fig 3a, 3b).

 

Preliminary Phytochemical Screening:

Physico-chemical studies: Ash value of a drug gives an idea of the earthy matter or the inorganic composition and other impurities present along with the drug. Extractive values are primarily useful for the determination of exhausted or adulterated drugs. The water soluble extractive was high in leaves of P.alba. The results of physico-chemical constants of the drug powder are presented in (Table – 1).

 

Figure: 6

 

Fluorescence Analysis of Drug Powder:

Fluorescence analysis of drug powder treated with acids was studied at day light and the observations are presented in (Table 2).

 

Quantitative microscopical data:

Pertaining to the stomatal index, stomatal number, veinislet number and veinlet termination number features are given in (Table 3).

Preliminary phytochemical screening:

Revealed the presence of Terpenoids, flavonoids, Alkaloids, Glycosides and Phytosteroids (Table - 4).

 

HPTLC Profile:

The Rf values of these extracts shows some of the compounds are matching. This shows clearly the presence of some of similar compounds in hexane, chloroform, ethyl acetate and methanol extracts. This provides an interesting observation that which extracts shows major number of compounds and marker compounds (Figure 4-7).

 

 

CONCLUSION:

As there is no pharmacognostic / anatomical work on record of this much valued traditional drug, the present work was taken up with a view to lay down standards which could be useful to detect the authenticity of this medicinally useful plant. In other words, the pharmacognostic features examined in the present study may serve as tool for identification of the plant for validation of the raw material and for standardization of its formulations.

 


Table : 4 Preliminary Phytochemical Test for Extracts

Extracts

Carbohy drates

Phytoste roids

Alkal oids

Flavon oids

Terpen oids

Glyco sids

Sapo nins

Gum

Mucilage

Hexane

-

+

-

+

-

-

-

-

CHCl3

-

+

+

+

+

-

-

-

Ethylacetate

-

+

+

+

+

+

-

-

Methanol

-

+

+

+

+

+

-

-

-  Negative, + Positive


 

Figure: 7

 

References:

1.        Henry AN, Kumeri GR, Chitra V.  Flora of Tamil Nadu, India. 1987; pp.78.

2.        Chopra RN, Nayar SL and Chopra IL. Glossary of Medicinal Plants. C.S.I.R., New Delhi. 1956; pp.198.

3.        Hartwell JL. Plants used against cancer (A survey) Quarterman Publications, Inc. Lawrence, Massachu setts. 1982; pp.408.

4.        Kirtikar KR and Basu BD. Indian Medicinal plants, International Book Distributors, Dehradun. 1935; pp 1548.

5.        Asolkar LV, Kakkar KK, Chakre OJ.  Second Supplement to Glossary of Indian medicinal plants with active principles. 1992; pp.173.

6.        Nargis Akthar, Abdul Malik, Saminanoor Ali. A New anti bacterial triterpenoid from Plumeria alba. Fitoterapia 1993; 2: 162-166.

7.        Rengaswami S, Venkatarao E. Chemical Components of Plumeria alba. Proc. Indian Acad.Sci 1960; 52(A): 173-181.

8.        Coppen, JJW and Cobb AL. Phytochemistry 1983; 22(1): 125.

9.        Brain KR and Turner TD. he Practical Evaluation of Phytopharmaceuticals, Wright – Scientechnica, Bristol 1975; pp. 4 -9.

10.     Johansen DA. Plant Microtechnique, McGraw Hill, New York, 1940; pp.182.

11.     Wallis TE. Text book of Pharmacognosy fifth edition, CBS publishers, New Delhi, India, 1985; pp.652.

12.     Trease GE.  A text book of Pharmacognosy, Bailliere, Tindall, London, 1961; pp. 34.

13.     Evans WC. Trease and Evan’s Pharmacognosy, Fourteenth edition, WB Saunders Co., Ltd., London, 1996. pp. 1 -40.

14.     Indian Pharmacopoeia, 4th edn., Vol. II, Government of India, Ministry of Health and Welfare, Controller of Publications, New Delhi, 1996; pp. A53-A54.

15.     WHO/PHARM/92.559/rev.1., Quality Control Methods for Medicinal Plant Materials, Organisation Mondiale De La Sante, Geneva, 1992; pp. 9, 22-34.

16.     Chase CR and Pratt RJ.  Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification.  J. Am. Pharmacol. Assoc. 1949; 38, 32.

17.     Kokate CK.  Practical Pharmacognosy, 1st edn, Vallabh Prakashan, New Delhi, 1986. pp. 15-30.

18.     Harborne JB.  Methods of extraction and isolation. in: Phytochemical Methods, Chapman & Hall, London, 1998. pp. 60 -66.

 

 

 

 

 

Received on 08.11.2008       Modified on 10.11.2008

Accepted on 12.12.2008      © RJPT All right reserved

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 496-501