Pharmacognostical and Phytochemical Studies on Leaves of Cinchona officinalis

 

Pradeepa R

Department of Pharmacognosy, College of Pharmacy, Madurai Medical College, Madurai, India.

Faculty of Pharmacy, KMCH College of Pharmacy, Coimbatore, India.

*Corresponding Author E-mail: Pharmacy_1791993@yahoo.com

 

ABSTRACT:

Pharmacognostical and phytochemical studies were carried out on Cinchona officinalis leaves belong to the Family Rubiaceae. It is one of the important traditional medicines. Different parts of this plant used traditionally for the treatment of malaria, typhoid, irregularity of heart, mild central nervous system depresents, local anaesthetic, hypotensive, antispasmodic action on smooth muscle, cardiac arrhythmia, cardiac depressant particularly in auricular fibrillation.The present investigation deals with the leaves of C.officinalis morphology parameters and microscopical studies such as Transverse section, Scanning Electron Microscope, Leaf constants, physicochemical, and elemental analysis by EDS. The shade dried leaf was powdered which is followed by the florescence analysis was carried out on powdered leaf. Standardization of the plant is done with WHO parameters. The leaf was extracted with ethanol in Soxhlet apparatus, the extract was collected and preserved in a vaccumdessicator. Phytochemical studies were carried out by using ethanol extract. The present study include the macroscopy, microscopy, phytochemical evaluation and elemental analysis are effective and economical botanical identity which can be supportive to standardization and authentication this plant material.

 

KEYWORDS: Cinchona officinalis, Rubiaceae, Pharmacognostical standardization, Phytochemical analysis.

 

 


INTRODUCTION:

C.officinalis Linn (CROWN or LOXA BARK) is a tree 6-15cm in height with rough brown bark, having pale yellow markings, inside and dull white ones outside, 7-15cm long. Ovate-lanceolate leaves with reddish green petioles, red flowers in coryliform cymes, oblong 12-20mm long capsules and elliptic winged seeds. It thrives well at higher elevations up to 2,600m in South India.

 

 

Quinine is most important alkaloid found in cinchona plant. Several alkaloids isolated from cinchona such as quinidine, cinchonine, cinchonidine, javanine, quinamine, salts of quinic and cinchotannic acids. Increase in the production of quinine and quinidine effected in tissue culture techniques4. The present investigation highlights the pharmacognostical studies involve macroscopy, microscopy, leaf constants, physicochemical parameters and phytochemical screening of the leaves of Cinchona officinalis L. The research on development of herbal products from this plant is required to be initiated immediately for exploring the unique potential of this crop which would also minimize the menacing wastage especially the leaves.

 

Fig . 1 Leaves                                         Fig .2 Cinchona officinalis

 

MATERIAL AND METHODS:

PLANT COLLECTION AND AUTHENTIFICATION AND STORAGE:

Leaves of the plant C.officinalis selected for our study was collected from ValparaiTaluk, Coimbatore District, Tamilnadu, India during the month of July 2016 and wasauthenticated by Dr.Stephen, Department of Botany, American college, Madurai and Dr. Sasikala, Director (Retd) of Siddha Central Research Institute, Arumbakkam, Chennai. The leaves were collected and shade dried. It was powdered in a mixer. The powder wassieved in a No.60 sieve and kept in a well closed container in a dry place it was used in pharmacognostical and phytochemical studies

 

MORPHOLOGY:

Fresh leaves of C. officinalis were collected, the various macroscopical characters vizsize, shape, colour, margin, texture, arrangement were observed and evaluated as per standard WHO guidelines [15].

 

Microscopy:

Care was taken to select healthy plants and for normal organs. Leaf, Petiole specimens were collected from a healthy plant by making a cut with petioles. The materials were cut into pieces and immediately immersed in fixative fluid FAA (Formalin–5ml+Acetic acid–5ml+70% Ethyl alcohol–90ml). After 24 hours of fixing, the specimens were dehydrated with graded series of ethyl alcohol and tertiary-butyl alcohol by Sass, 1940. After dehydration, the shavings of paraffin wax were added to the vial containing the plant material with pure TBA. The paraffin shavings are added every 30mins at about 40-45ºC four or five times. Then the vials were filled with wax without damaging the tissues. The vial filled with wax is kept open in warm condition to evaporate all TBA, leaving the specimen in pure molten wax. The specimen filled with pure molten wax for 2 or 3 times by decanting the old wax every time [10-20].

 

Scanning Electron Microscopy (SEM):

Sample for SEM analysis were mounted on the specimen stub using carbon adhesive sheet. Small sample were mounted with I sq. cm glass slide and kept in carbon adhesive sheet.Samples were coated with gold to a thickness of 100 AO using hitachi vacuum evaporator. Coated sample were analysed in a Hitachi Scanning electron Microscope 3000 H model. The micrograph from SEM shows the best possible structural details of the specimens. In addition, shows the unsuspected details and any undescribed characters [8-18].

 

Leaf constants:

Clear the piece of leaf by boiling with chloral hydrate solution and temporary preparation made by using glycerine is a mounting reagent. Study of the vein islet number, vein terminal number, stomatal number and stomatal index were determined on cleared leaf using standard procedures [9-12].

 

Physicochemical parameters:

The study of ash values, total ash, water soluble ash, acid insoluble ash, extractive value, loss on drying were determined by using air dried powder of the leaf as per the official method [2-5].

 

Preliminary Phytochemical studies:

The leaf was extract with ethanol in a Soxhlet apparatus at 60C in 3 hours. This extract was subjected to various phytochemical analysis by using standard test procedure [1-13].

 

Elemental analysis by Energy Dispersive Spectrum (EDS):

Scanning Electron Microscope model with an EDS microanalysis attachment was used to study the microscopical characters and elementals present [8].

 

Florescence analysis:

Powdered leaf material Of C.officinalis was subjected to analysis under UV light after treatment with various chemical and organic reagents like Ethanol, Ethyl acetate, Chloroform, Water, 50% sulphuric acid, 10% sodium hydroxide, 50% nitric acid and dried leaf powder [7].

 

RESULT AND DISCUSSION:

ORGANOLEPTIC CHARACTERS FOR POWDER

Nature:                 Coarse

Colour :                Brown

Odour :                 Pleasant

Taste                     Aromatic

 

MACROSCOPY

Shape:                  Simple, opposite, varying from broadly ovate to lanceolate

Size:                     Length 10-12.5cm long, Width 18cm

Colour:                 Deep green

Margin:                Recurved margin

Apex:                   Rounded

Base:                    Broadly to narrowly cuneate

Petiole:                 Smooth andcylindrical

 

MICROSCOPY:

T. S of Leaf through midrib:

Transverse section of midrib shows a hump on the adaxial side and a big convexity on theabaxial side. Epidermis is made up of small rectangular cells and covered by a thick cuticle. (Fig 3.1)

 

 

Fig 3.1 T.S of Leaf through midrib

 

AdE–Adaxial Epidermis, AbE–Abaxial Epidermis, Col–Collenchyma, Par–Parenchyma,

 

Pal–Pallisade cells, SpP–Spongy Parenchyma, LV–Lateral Vein, Ph–Phloem, Xy–Xylem, GT–Ground Tissue.

 

T.S of Midrib–Enlarged portion:

The vascular bundle is large, deeply crescentic but with 2 rows of accessory strands between the ends of the crescent. (Fig 3.2)

 

 

Fig 3.2 T.S of Midrib enlarged portion

 

T.S of Midrib- Upper portion

The hypodermal region is composed of 3 or 4 rows of collenchyma cells. (Fig 3.3)

 

 

Fig 3.3 T.S of Midrib Upper portion

 

T.S of Midrib–Lower portion:

The ground tissue is parenchymatous and made up of circular to oval, closely packed parenchyma cells with small intercellular spaces. (Fig 3.4)

 

 

Fig 3.4 T.S of Midrib Lower portion

 

Lamina:

Leaf is dorsiventral and hypostomatic in nature. Epidermis is made up of small rectangular cells covered with thick cuticle. Hypodermis is composed of large parenchyma cells.The mesophyll is differentiated into palisade and spongy tissues. The upper palisade tissue is made up of 2 layered columnar closely arranged cells. The lower spongy tissue is composed of6-8 layers of round to oval parenchyma cells with small intercellular spaces. Externally to the lateral veins are noticed.(Fig 4).

 

 

Fig 4 T.S of Lamina

 

AdE-Adaxial Epidermis, Pal-Palisade, LV-Lateral Vein, SpP-Spongy Parenchyma,

AbE- Abaxial Epidermis

 

Epidermis in surface view:

Adaxial epidermal cells are nearly polygonal with slightly wavy walls and devoid of stomata. Abaxial epidermal cells have wavy contour. Paracytic or rubiaceous stomata are confined to the abaxial surface. (Fig 5)

 

 

Fig 5 Epidermis in surface view

Scanning Electron Miscroscopy (SEM):

The abaxial leaf surface was studied using SEM. It showed the presence of paracytic or Rubiaceous stomata. The abaxial epidermal cell walls are wavy (Fig 6).

 

 

Fig 6 Paracytic stomata enlarged view

 

T. S of PETIOLE:

Transverse section of petiole presents a circular outline with two short wings on theadaxial side. Epidermis is single layered and covered by a thick cuticle. In transverse section through the distal end of petiole exhibits a deeply crescenticto cylindrical median bundle, enclosing a few small medullary strands. The vascular bundle is accompanied with two small accessory strands towards the wing, on the adaxial side.Secretory cells are present in the parenchymatous tissue. The characteristic feature is the presence of elongated mucilage sacs situated externally to the phloem (Fig 7.1).

 

 

Fig 7.1 T.s of Petiole

 

ED–Epidermis, Col-Collenchyma, Par-Parenchyma, VB-Vascular Bundle, MS- Musilage Sacs, SC-Secretory Cells

 

T.S of Petiole-cortex:

The cortex is broad and divided into 2 regions. The outer region is made up of 3 or 4 rows of collenchyma cells and inner 6-8 rows of thick walled parenchyma cells. (Fig 7.2)

 

 

Fig 7.2 T.S of Petiole Cortex

 

T.S of Petiole–Midrib:

The large vascular bundle is situated in the centre. It is deeply crescentic but with 2 rows of accessory strands between the ends of the crescent. Vessels are circular and arranged in radial multiples of 3-6 (Fig 7.3)

 

 

Fig 7.3 T.S of Petiole Midrib

 

LEAF CONSTANTS:

Table 1: Leaf constant of Cinchona officinalis

S.NO

PARAMETERS

VALUES in Sqmm

1

Stomatal number (lower epidermis)

10-13

2

Stomatal Index (lower epidermis)

6–8

3

Vein islet number

5–8

4

Vein termination number

4-7

 

PHYSIOCHEMICAL PARAMETERS:

Table 2: Physiochemical parameters of Cinchona officinalis leaves

S.NO

PARAMETERS

VALUES

I

ASH VALUE

1

Total ash

6.79

2

Acid insoluble ash

1.18

3

Water soluble ash

1.31

II

EXTRACTIVE VALUE

1

Alcohol soluble extractive

4.3

2

Water soluble extractive

7.2

III

Loss on drying

1.38

 

PRELIMINARY PHYTOCHEMICAL STUDIES:

Table 3: Phytochemical analysis of ethanolic extract of Cinchona officinalis leave

S.NO

TEST OF CONSTITUENTS

Ethanol

1

Alkaloids

+

2

Carbohydrates

+

3

Glycosides

-

4

Sterols

+

5

Saponins

+

6

Tannins

 

7

Proteins and free amino acids

+

8

Mucilage

-

9

Terpenoids

+

10

Flavonoids

+

11

Volatile oil

-

12

Fixed oil

-

 

Elemental analysis by EDS:

Trace elements are considered the inorganic switches in various medicinal systems. This concept has gained ground in Ayurveda and the traditional Indian medicinal systems. Mineral contentsof various medicinal plants correlated with their therapeutic action by numerous studies.

 

The weight percentage of mineral were present Carbon (62.04%), Nitrogen (1.78%), Oxygen (34.90%), Sodium (0.16%), Magnesium (0.24%), Silicon (0.13%) , chloride (o.12%), Potassium (0.17%), Calcium (0.45%) (Fig 7.4)


 

FLORESCENCE ANALYSIS:

Table 4: The fluorescence analysis of the leaf powder of Cinchona officinalis.

No

Treatment

Visible light

UV 254nm

UV 365nm

1

Powder as such

Pale green

Green

Dark brown

2

Powder+Ethanol

Brown

Green

Brown

3

Powder+Ethylacetate

Brownish yellow

Dark green

Dark brownish black

4

Powder+Chloroform

Brownish yellow

Dark green

Dark brownish black

5

Powder+Water

Brown

Dark green

Black

6

Powder+50% Sulphuric acid

Dark brown

Green

Black

7

Powder+10%Sodiumhydroxide

Fluorescence yellow

Dark green

Bluish black

8

Powder+50%Nitric acid

Yellowish brown

Dark green

Brownish black

9

Powder+50%Hydrochloric acid

Brown

Dark green

Black

 


 

Fig 7.4  Elemental analysis of C.officinalis leave

 

CONCLUSION:

Morphological and microscopical examination and characterization of medicinal plants have always been accorded due credentials in the pharmacological studies. There was no detailed pharmacognostical work has been carried out including botanical identity based on micro morphology in this leaves of this plant. The application of morphological studies in drug analysis is pertinent in the field of crude drug authentication. It was studied for the leaf. Interpretation of the morphological characteristics based on different parameters, for the plant organs give a guideline for the diagnosis of the original plant and its adulterants. Microscopic techniques help to magnify the fine structure of minute objects and there by confirm the structural details of the plant drug. The present study of macroscopy, microscopy, leaf constants, physiochemical, phytochemical, elemental anaylsis  and florescence analysis can be used to obtain full evidence for standardization and evaluation of herbal drugs.

 

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Received on 16.04.2018        Modified on 26.06.2018

Accepted on 13.07.2018     ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(3): 246-250.

DOI: 10.5958/0975-4385.2018.00040.7