phytochemical and pharmacognostic profile of the leaves of leptadenia...

Journal of Phytomedicine and Therapeutic 2018; 17(2): 189 – 213 96 www.jopat.gov.ng

JOPAT Vol. 17(2) 196 – 213, 2018. ISSN 2636 – 5448

PHYTOCHEMICAL AND PHARMACOGNOSTIC PROFILE OF THE LEAVES OF

LEPTADENIA HASTATA (PERS.) DECNE.

Ibrahim Jemilat A., 1, 2* Gwadabe Ummi S., 2 Fatokun Omolola T1 and Esievo Kevwe B1.

1 Department of Medicinal Plant Research and Traditional Medicine, National Institute for

Pharmaceutical Research and Development (NIPRD), P. M. B. 21, Garki, Abuja, Nigeria.

2 Department Of Biological Sciences, Faculty of Computing and Applied Sciences, Baze

University, Abuja, Nigeria.

ABSTRACT

Objective: This study was designed to evaluate the phytochemical constituents and

pharmacognostic parameters, which could be helpful to ensure the purity and safety of Leptadenia

hastata.

Method: Phytochemical analysis and pharmacognostic studies including microscopy, chemo

microscopy and proximate analysis were conducted.

Result: Leaf epidermal study reveals polygonal cell and irregular cell shape on adaxial and abaxial

surfaces, respectively with striated abaxial cells and anomocytic stomata type. Leaf transverse

section shows two layers of palisade cells, abundant oil globule, starch grains and multicellular

trichomes on both abaxial and adaxial epidermises. Quantitative leaf microscopy reveals stomata

number (16 ±1.0), stomata index (14.97), vein islet number (56.3 ±0.3) and vein termination

number (4.7 ±0.7). Powdered microscopy of leaf revealed the presence of parenchyma cells, spiral

xylem vessels and epidermal cells with anomocytic stomata. Chemomicroscopy indicated

presence of lignin, starch, protein, oil, cellulose and calcium oxalate crystals. The physicochemical

parameters evaluated were: Ash value 13.61%, water and alcohol extractive values 30.125% and

15.23 %, moisture content 6.95%. Preliminary phytochemical investigations showed the presence

of tannins, flavonoids and saponins.

Conclusion: The pharmacognostical and preliminary phytochemical characters observed in the

leaf of Leptadenia hastata could help in the plant identification and provide clues to its

standardization. The information could also be useful in the development of the monograph of the

plant.

Keywords: Leptadenia hastata, pharmacognostic studies, proximate analysis, phytochemical

analysis

Correspondence email- [email protected] ©2017The authors. This work is licensed under the Creative Commons Attribution 4.0 International License

Ibrahim et al

Journal of Phytomedicine and Therapeutic 2018; 17(2): 189 – 213 Page 197 www.jopat.gov.ng

Introduction

Leptadenia hastata (Pers.) Decne.,

(Asclepidiaceae) is a leafy vegetable plant

found in nature as a wild plant. It is a climber

with pale white, soft and grooved stem.

Leave is simple, acuminate and ovate shaped

and exude white latex when crushed. Flowers

are cream or yellowish green and fruits are

dehiscent, two-valved releasing cotton

winged seeds [8]. It is widely distributed in

tropical Africa including northern Nigeria

[15]. The plant is locally known in Nigeria as

Yadiya, Dan- bakwa (Hausa) Kalimbo, Njera

(Kanuri) Isanaje-igbo, Ogbo funfun

(Yorubas) [7, 13].

In Nigeria, the plant is used as a spice and in

preparation of sauces [14] and considered as

a famine food in some parts of Africa [12, 15,

21]. Leptadenia hastata is used in Nigeria

ethnomedicine to manage hypertension,

catarrh and skin diseases [9], gonorrhea and

stomach-ache in children [23].

Pharmacological studies have shown that the

plant has antimicrobial [2], Anti-androgenic

[5], Anti – inflammatory and wound healing

[20] and anti-diabetic activities [6].

The major secondary metabolites present in

L. hastata are chemical constituents isolated

from L. hastata, which include lutein, lupeol,

β- carotene, kidjolanin, cynanforid and

gagaminin [4, 6,19].

The increased use of herbal drugs and

concern over their safety and efficacy has

certainly augmented the need for their

standardization. World health organization

(WHO) set up some guidelines for

standardization of crude drugs which include

morphological, microscopic, physical,

chemical and biological observation. These

standards are crucial in establishing the

quality, identity and purity of crude drugs [1,

17]. Pharmacognostic standards must be set

for every crude drug to be included in an

herbal pharmacopoeia.

The aim of this study was to evaluate the

pharmacognostical parameters of

Leptadenia hastata towards standardization,

monograph development on the plant and

for quality control purposes.

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Figure 1: Photographs of Leptadenia hastata

Key: A: plant showing ovate leaf shape; B: Plant in its habitat showing it habit; C: fruits

Materials and Method

Chemicals, reagents and solvents

All chemicals, reagents and solvents used

during the experimentation were of analytical

grade.

Collection

The fresh leaves of Leptadenia hastata were

collected on 18th of October 2017, from

Airport road Jiwa Abuja. The plant was

identified and authenticated in the herbarium

of National Institute for Pharmaceutical

Research and Development, Abuja, by Dr.

Ibrahim. Voucher specimen was deposited in

the same herbarium with voucher no:

NIPRD/H/6889.

Sample Preparation

Collected fresh leaves were preserved in 50%

ethanol for microscopic study (transverse

sectioning of leaves), while some were dried

in between old newspaper for epidermal

peeling. Some leaves of the plant were also

air dried for a week and powdered using

electrical blender. The powdered sample was

stored in a polythene bags for further use.

Macroscopic / Organoleptic Study

A

B

C

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The leaves were subjected to macroscopic

analysis viz. organoleptic characteristics such

as appearance, taste, colour, odour, shape,

texture, fracture, etc. of the drug. These

parameters are considered to be quite useful

in quality control of the crude drug and were

evaluated as required by WHO guidelines [1,

24].

Epidermal Leaf Microscopy study

Microscopic analysis was carried out on the

upper and lower epidermal surfaces of the

leaf and pulverized leaf samples. The method

of Jemilat et al [16] was used to prepare

epidermal surfaces of the leaf. About 5 mm2

– 1 cm2 leaf fragments were obtained from

the standard median portion of the leaf and

macerated in concentrated nitric acid in petri-

dish for a period of 18 h The appearance of

air bubbles indicated the readiness of the

epidermises to be separated. The fragments

were transferred into water in a petri-dish

with a pair of forceps. The upper, lower

epidermises and mesophyll were separated

and cleaned using forceps and carmel hair

brush. Epidermal peels were stained with

safranin and mounted on a slide with

glycerol. Subsequently, some leaf samples

were cut and soaked in sodium hypochlorite

for clearing to study the venation pattern.

Transverse section of leaves

Sections were manually obtained by

sectioning with a razor blade. The sections

were cleared for some minutes in 3.5%

hypochlorite solution, washed with water and

stained with sudan IV solution. These were

mounted on a slide with glycerol.

Quantitative microscopy

Quantitative examinations such as vein-islet

number, vein-termination number, palisade

ratio, stomatal number and stomatal index

were carried out using standard methods [16].

Chemo-microscopic Studies

Chemo-microscopic studies of the pulverized

leaf was done using reagents and stains like

iodine, concentrated Sulphuric acid,

concentrated hydrochloric acid, ferric

chloride, Sudan III, ruthenium red and

phloroglucinol with conc. HCl (1:1) to test

for the presence of various parameters [1].

Microphotography

Photomicrographs of different sections were

taken using Leica CME microscope with

digital microscope eyepiece attachment and

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Photo Explorer 8.0 SE Basic software at

different magnifications (x100 and x400).

Phytochemical Screening

Standard screening tests of the powdered

leaves sample for the presence of secondary

metabolites such as phenolic compound,

tannins, saponins, flavonoids, cardiac

glycosides, and anthraquinones were done

according to standard methods [22].

Chromatographic fingerprinting

Analytical TLC was done on silica gel plate

. The plant material was extracted

successively with hexane, ethyl acetate and

methanol. The plate was developed, after

spotting the extracts at the origin using

solvent system hexane and ethyl acetate, ratio

4:3. Detection was done in daylight, under

UV366nm and with 10% aqueous H2SO4

spray reagent. Plates were dried at 100°C

after spraying. The different retardation

factors (Rf) of each spot were calculated [18].

Statistical analysis

The data obtained were expressed as mean ±

SEM (standard error of mean), and n

represents the number of replicates in an

experiment.

RESULTS

Macroscopic / organoleptic

characteristics

Leaves of L. hastata are simple, opposite in

arrangement, deciduous and stipulate. Leaf is

ovate with acuminate apex and entire blade

showing parallel venation. Upper surfaces

are dark green, while lower surfaces are light

green in color with a bristle texture (Figure

1). The powdered leaf material is green in

color, with characteristic odour and tasteless.

Microscopic studies

The adaxial epidermis shows abundant non-

glandular trichomes. Epidermal cells are

polygonal in shape, striations were present

but no stomata were observed. The abaxial

epidermis also shows abundant multicellular

trichomes epidermal cells are elongated,

irregularly shaped with wavy outline,

abundant anomocytic stomata type.

Epidermal cells are striated; striations are

more on the subsidiary cells and prismatic

crystals were seen (Fig 2 & 3). The

quantitative leaf microscopy measurements

are shown in Table 1.

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The T/S of the leaf showed a layer of

epidermal cells on both adaxial and abaxial

surfaces, two layers of palisade cells, spongy

mesophyll, abundant oil globules scattered

on the palisade cells. Druses (crystals) are

abundant, especially on the epidermal cells

and multicellular trichomes. Spiral xylem

vessel and large vascular bundle were seen at

the midrib. Cells closer to the palisade cell

also contained starch grains (Fig 4, 5 & 6).

The powdered leaves sample showed

fragment of parenchyma tissues, fragment of

epidermal cells with stomata, different types

of multicellular trichomes, fragments of

spiral xylem vessel, abundant starch grains

and oil globules and prismatic crystals (Fig

7 & 8).

The result of the chemo microscopic

evaluation is shown in Table 2.

Physicochemical parameters

Determination of physicochemical

parameters of a crude drug is essential as it

helps in identification and estimation of

mishandling, adulteration and also in setting

of proper standards. Physicochemical

parameters viz ash values, extractive values,

and moisture content were investigated and

the results are presented in Table 3.

Phytochemical Analysis and Thin Layer

Chromatography (TLC)

The result of phytochemical screening is

presented in Table 4. The Retardation Factor

(Rf) values of spot detected from TLC

analysis of the three extracts (hexane, ethyl

acetate, and methanol extracts) developed in

the solvent system of Hexane: ethyl acetate

(4:3) are presented in Table 5.

Discussion

Some important diagnostic characters that

might be useful in determining authenticity

and identifying adulteration of the crude drug

were observed in this study. Macroscopy

characters, organoleptic and epidermal

characteristic of leaf of Leptadenia hastata

especially the striated abaxial epidermal and

subsidiary cells, prismatic crystals and

different multicellular trichomes are

diagnostic features for the plant. The stomatal

index of 14.97 per mm², vein islet number of

56.3 per mm², and vein islet termination of

4.7 per mm², number of epidermal cells

(adaxial) 90 per mm², number of epidermal

cells (abaxial) 91per mm²provide valuable

information regarding leaf constants which

are useful parameters in standardization.

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Abundance of oil globules scattered on the

palisade cells, abundant druses found all over

the cell especially on the trichomes seen on

transverse section of the leaves are also

diagnostic and can be used to determined

authenticity of the plant even when in

fragments.

Ash values are reliable tools in detecting

adulteration and in establishing the purity of

crude drugs [1]. The ash value of 13.6%

shows that the plants contain low amount of

inorganic and other impurities present along

with the plant. Moisture content of 6.95%

falls within the recommended range of 8 –

14% for vegetable drug - an indication that

the plant can be stored for a long period with

less probability of microbial attack. High

water content promotes the growth of

microorganisms leading to degradation and

spoilage. The report of Fai et al [11] showed

differences in moisture content and ash

content in samples collected in Abuja (North

central) and Gombe state (North East),

Nigeria. The observed differences could be

due to geographical location.

Phytochemical investigations indicated the

presence of carbohydrate, Tannins,

flavonoids, and saponins. Phenols, tannins,

flavonoids, and saponins were also present in

the leaves of Leptadenia hastata collected

from northern part of the country [6].

Presence of these metabolites might be

responsible for various pharmacological

activities of the plant. Tannins have anti-

diarrheal activity and have also been reported

in the treatment of sexually transmitted

diseases; saponins for gastrointestinal

infection; Flavonoids are free radical

scavengers and therefore useful in

management of inflammatory diseases [3, 25,

26]. The alcohol extractive value of 15.3 and

water extractive value of 30.1 signify the

amount of organic constituents presents.

Extractive values are primarily useful for the

determination and evaluation of the chemical

constituents present in the crude drug and

they also help in the estimation of specific

constituents soluble in particular solvents

[10]. Water soluble extractive value of 30.1%

was two times higher than alcohol soluble

extractive value of 15.2% which means

greater yield was obtained using the aqueous

medium than the organic medium.

The thin layer chromatography of various

extracts showed spots which have different

Rf values. Rf value of various spots provide

valuable clue regarding their polarity and

selection of solvents for separation and the

profile gives a preliminary view of the

secondary metabolites that are present. The

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various spots observed on TLC can be used

as finger-prints in the identification of L.

hastata.

Conclusion

The pharmacognostic evaluation of L.

hastata leaf is being reported for the first time

and results from this study have provided

information on the morphological and

anatomical features, and the physicochemical

parameters of L. hastata leaf. These

parameters can be used for identification and

quality control of the plant drug and provide

information, which may be useful in

monograph development on the plant.

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Triterpenoids from Leptadenia

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C., Tsin, AT., Muhamadu I, et al.

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M., Guissou, I.P and Sawadogo, L

(2005). Ecological distribution,

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Fig 2 A: Upper Epidermal Layer of Leptadenia hastate, B: Lower Epidermal Layer of Leptadenia

hastate, C: Leaf Surface of Leptadenia hastata showing Vein Islet and Vein Termination

Key: PE: polygonal epidermal cells, IS: irregular shape cells, ST: striations, AS: anomocytic

stomata type, TB: Trichome base, VI: Vein Islet; VT: Vein Termination

AS

ST

TB

IS B

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Fig 3: Trichomes and Prismatic crystals on Epidermal Layers of Leptadenia hastata

Key: TC: Trichomes; PC: Prismatic crystals

Fig 4: Transverse Sections of Leaves of Leptadenia hastata

Key: MR: Midrib; PC: Palisade cells; TR: Trichomes; Pl: Phloem; Xy: Xylem

PC TR

Ph

Xy

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Fig 5: Transverse Sections of Leaves of Leptadenia hastata showing spiral xylem vessels, 2

layered palisade cells and striated trichomes

Key: XV: Xylem Vessels; PC: Palisade Cells; TR: Striated Trichomes; EP: Epidermal

Cells; SG: Starch grains

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Fig 6: Transverse Sections of Leaves of Leptadenia hastata showing mesophyll and palisade

layers

Key: PC: 2 layers of Palisade Cells; MC: Mesophyll cells; OG: Oil globules

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Fig 7: Microscopy of powdered leaves of Leptadenia hastata

Key: PT: Parenchyma tissue; EP: Epidermal cell; ST: Stomata; Star: Starch grains; OG:

Oil globules; XY: spiral xylem vessel

Fig 8: Microscopy of powdered leaves of Leptadenia hastata showing different Trichome

Types (TR)

TR

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Table 1: Quantitative Leaf Microscopy of Leptadenia hastata

* - n = 12, *1 - n =3

Table 2: Chemo-microscopy of the Powdered Leaves of Leptadenia hastata

Test Inference

Lignin +

Cellulose +

Tannins +

Starch +

Oil +

Protein +

Crystal +

Key: + = present; - = absent

Parameters Range (per mm2) Mean ± SEM

Number of adaxial Epidermal cells* 79 – 100 90 ± 1.8

Number of abaxial Epidermal cells* 80 – 103 91 ± 2.5

Stomata number (adaxial) absent absent

Stomata number (adaxial)* 10 – 21 16 ± 1.0

Stomatal index (abaxial)*1 14.97

Vein islet number 56 – 57 56.3 ± 0.3

Vein termination number*1 4 – 6 4.7 ± 0.7

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Table 3: physiochemical parameters of the leaves of Leptadenia hastate

Test Result (%)

Moisture content 6.95

Total ash 13.61

Water extractive value 30.13

Alcohol extractive value 15.23

Table 4: Phytochemical Screening of the Powdered Leaf of Leptadenia hastate

Key: + = Positive; - = Negative

Test Inference

Carbohydrate +

Saponins +

Tannins +

Flavonoids +

Terpenes _

Steroils _

Glycosides _

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Table 5: Retardation Factor (Rf) of Components of Organic extracts of Leaves of

Leptadenia hastata in Solvent System Hexane:Ethylacetate (4:3)

Extract Rf Daylight UV366nm 10% v/v aq

H2SO4

Hexane 0.48

0.67

0.85

0.86

0.89

0.91

Light yellow

-

Green

Yellow

-

-

-

-

Pink

-

Pink

Brown

Light brown

Brown

Brown

Brown

Brown

Ethlyacetate 0.41

0.46

0.52

0.59

0.78

0.84

0.91

Light yellow

Light yellow

Green

Light yellow

Green

Darkgreen

yellow

-

Pink

Green

Pink

Pink

Green

-

Brown

Brown

Brown

Brown

Brown

Green

Brown

Methanol 0.46

0.77

0.85

0.77

Light yellow

-

-

-

-

Pink

-

-

Brown

Brown

Brown

Brown

phytochemical and pharmacognostic profile of the leaves of leptadenia...

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