International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 4(6) pp. 149-157, June, 2013
Available online http://www.interesjournals.org/IRJPS
Copyright © 2013 International Research Journals
Full Length Research Paper
Comparative evaluation of the Pharmacognostic,
Phytochemical parameters and Microscopic studies of
the leaves of Gardenia erubescens and Gardenia
ternifolia (Family rubiaceae)
*Jemilat Aliyu Ibrahim, Jephthah O. Odiba and Oluyemisi Folashade Kunle
Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and
Development (NIPRD), PMB 21, Garki, Abuja, Nigeria
*Corresponding Author Email: sadiqoyene@yahoo.com
Abstract
Preliminary pharmacognostic, phytochemical and microscopic analyses were carried out on the leaves
of Gardenia erubescens Stapf and Hutch and Gardenia ternifolia Schum and Thonn., Family Rubiaceae.
The moisture content was 9.1% and 10.7%, ash value 5.6% and 5.3%, and acid-soluble ash value 2.8%
and 2.4% respectively. Phytochemical screening revealed the presence of nine secondary metabolites
while Thin Layer Chromatography (TLC) revealed several spots for the hexane, ethylacetate and
methanol extracts. The study reveals microscopic characters that are useful as diagnostic parameters
for the two Gardenia species. Information obtained from this study is important in establishing
diagnostic indices for identification, standardization, and also in monograph development of the plants
which have many ethno medicinal uses.
Keywords: Microscopy, Gardenia erubescens, Gardenia ternifolia.
INTRODUCTION
Gardenia erubescens Stapf and Hutch and Gardenia
ternifolia Schum and Thonn belong to the family
Rubiaceae. Gardenia erubescens is a shrub or small tree
of about 3m high, branching from near the base and it is
found in the savannah or savannah wood land (Burkil,
1997). The plant is commonly distributed in Senegal to
Northern Nigeria, Sudan and Uganda (Burkil, 1997). The
leaves are broadly obovate with rounded apex and
cunneate base. The fruits of G. erubescens are leathery
and fleshy, not fibrous or ribbed as in G. ternifolia (Irvine,
1961). Gardenia ternifolia is also a shrub or small tree of
about 5m high, distributed across tropical Africa e.g Mali,
Ivory-coast, Nigeria, Chad, Sudan and Senegal (Burkil,
1997). The leaves are oblanceolate to obovate, leathery
with wavy margin. Fruits are oblong to elliptical with
fibrous pericarp (Irvine, 1961). G. erubescens and G.
ternifolia are very important medicinally (Burkill, 1997). In
Senegal, leaves and root of G. erubescens are added in
formulation for both external and internal treatment of
syphilis (Kerharo and Adam, 1964), and in Gambia as
emetic purgative. Barks are used for gastro-intestinal
infection in children and also for treatment of gonorrhoea
(Dalziel, 1937 and Irvine, 1961). In Borno State, Nigeria,
the edible fruit of G. erubescens is used in treating
earache (Akinniyi and Sultanbawa, 1983) while the seed
is a source of black cosmetic known in Hausa as
“Katambiri” (Dalziel, 1937). Due to the yellow, hard,
compact and tough wood of G. erubescens, it is used in
making spoons, knife and handles of small implements
(Dalziel, 1937 and Irvine, 1961) Figure 1.
In Nigeria, the leaf infusion G. ternifolia is taken
internally to treat syphilis (Dalziel, 1937, Burkil, 1997); in
Gabon, it is applied on skin-itch (Walker, 1953). Also, the
leaves are used in bath and lotion to protect against
arrow poisoning in Ivory-Coast (Kerharo and Bouquet,
1950). The non-edible fruit of G. ternifolia is used as fish
poisoning; the bark promotes virility and root-bark is used
for dental caries. The sap is used as laxative to relieve
flatulence and dysentery; preparations containing roots
are used for female sterility, aphrodisiacs and poison
150 Int. Res. J. Plant Sci.
A
B
Figure 1. Picture of leaves and Fruit of Gardenia erubescens (A- Leaves; B- Fruit)
A
B
Figure 2. Picture of leaves and Fruit of Gardenia ternifolia (A- Leaves; B- Fruit)
antidote (Kerharo and Adam, 1962). Root boiled with
millet flour in Nigeria is used in the treatment of ‘Black
water’ fever and cough (Ainslie, 1937). Charred and
pulverised plant is put in palm wine with guinea grass and
Shea butter for severe constipation (Burkill, 1997). Some
of the traditional and medicinal uses of these species
have been confirmed by several authors (El Ghazali et
al., 1987; Farah et al., 2012) Figure 2.
Macrosopically, the fruits of Gardenia erubescens
and G. ternifolia can be distinguished easily. For
example, G. erubescens‘s fruit is fleshy and edible while
G. ternifolia fruit is not edible and the pericarp is hard and
fibrous. However, a clear distinction cannot be made of
the leaves of both plants especially when in fragments.
This study is aimed at establishing comparative
phytochemical, pharmacognostic and microscopic
parameters of the leaves of the two species in order to
prevent adulteration or substitution of one species for the
other. The findings will be useful in the standardization
and monograph development of the species.
MATERIALS AND METHOD
Collection of Plants
Gardenia erubescens and G. ternifolia plants were
collected from Chaza Village in Suleija LGA of Niger
st
State on the 21 of February, 2011 by Mall. Ibrahim
Muazzam. The plants were identified in the herbarium of
the National institute for Pharmaceutical Research and
Development, Abuja, Nigeria.
Plant sample processing
Fresh leaves were air-dried indoors for about eight days.
The leaves were powdered using mortar and pestle, but
later blended with electronic blending machine. The
powdered samples were stored in air tight containers for
the phytochemical analysis.
Microscopic examination
Epidermal layer preparation
Leaves of G. erubescens and G. ternifolia were cut at the
median portions. These were soaked in concentrated
nitric acid for about 24 hrs. The appearance of air
bubbles showed that the epidermises were ready to be
separated. The samples were then transferred to petri
dishes containing water and with the use of fine forceps
and dissecting needle the upper and lower epidermises
were separated. One set was stained with saffranin and
another one with Sudan IV and mounted on slides in
glycerol. The edges of the cover slip were sealed with
nail vanish to prevent dehydration. Subsequently, some
leaves were cut and soaked in sodium hypochlorite for
clearing.
Ibrahim et al. 151
Transverse section (T/S) of the leaves
Sections were manually obtained by sectioning with
razor blade. The sections were cleared for some minutes
in sodium hypochlorite solution. They were washed in
water and then stained with Sudan IV. These were
mounted on slides with glycerol and edges of the slides
were sealed with nail vanish to prevent dehydration. All
slides were observed under a light microscope and
photomicrographs taken using Olympus Microscope
Hyper Crystal LCD model No E-330 with camera
Olympus CX31 RTSF.
Determination of physicochemical constants
Determination of ash values, moisture content
and extractive values of the powdered leaves were
carried out according to standard methods (Sofowora,
2008; Evans, 2002; African Pharmacopeia, 1986).
Rf =
Distance moved by solute
Distance moved by solvent
RESULTS
Microscopic examination
Epidermal layers
Adaxial surface
The adaxial epidermal layer of G. erubescens showed
polygonal epidermal cells with straight anticlinal walls and
also hypodermal cells which were tetragonal and irregular
in shape (Plates 1a and b). The hypodermal cells were
larger in size than the epidermal cells (Plates 1a and b).
Oil globules were abundant on this surface. The adaxial
epidermal surface of G. ternifolia showed polygonal
epidermal cells with straight anticlinal wall; no
hypodermal cells were observed (Plates 1e and f).
Phytochemical analysis
Abaxial surfaces
The powdered leaves were screened for the presence of
secondary metabolites such as carbohydrate, tannins,
saponins,
anthraquinones,
flavonoids,
alkaloids,
glycosides, terpenes, resins, balsam, phenols and sterols.
The phytochemical screening was carried out following
standard methods (Sofowora, 2008; Evans, 2002).
Extraction
The powdered leaves of Gardenia erubescens and G.
ternifolia were extracted successively with n-hexane,
ethyl acetate and methanol for 24 hours. 20g of the
powdered leaf samples were macerated in 200ml of each
of the solvents successively. The extracts obtained were
concentrated using a rotary evaporator and brought to
dryness on water bath.
Thin- layer chromatography
Thin layer chromatography (TLC) of the extracts was
carried out to determine the number of components
present in each extract. TLC plates pre-coated with K5
silica gel were used. Spotting was done using capillary
tubes. Hexane, ethylacetate and methanol extracts were
developed in a solvent system of hexane and
ethylacetate, ratio 9:2. The methanol extract was later
developed in solvent system of methanol, chloroform and
ethylacetate in ratio 3:2:1. The plates were observed for
spots before and after spraying with dilute sulphuric acid
(Evans, 1996). The retardation factor (Rf) for each spot
was calculated using the formula:
Abaxial epidermal surface of G. erubescens was
characterised by abundant cyclocytic stomata, few
polygonal epidermal cells and crystals. Stomata were
numerous in number almost obscuring the view of the
epidermal cells (Plates 1c and d). The abaxial epidermal
surface of G. ternifolia also showed abundant anomocytic
stomata and irregular – shaped epidermal cells with
abundant oil globules (Plates 1g and h). Under lower
magnification, the abaxial surface of G. erubescens
showed the presence of abundant crystals while G.
ternifolia had abundant stomata (Plates 2a and b).
Transverse section
Gardenia erubescens
Dorsiventral leaf type with thick cuticle, one layer of
epidermal cells and one layer of continuous hypodermal
cells (Plate 3A). Hypodermal cells are larger than the
epidermal cells (Plate 3B). Three layers of palisade cells;
Idioblast cells containing rosette crystals abundant on the
palisade and spongy mesophyll cells (Plate 3B). Crystals
occasionally found in between the hypodermal cells. Oil
globules on epidermal cells, abundant spiral xylem
vessels and long fibers (Plate 3B).
Gardenia ternifolia
Dorsiventral leaf type with thin cuticle. One layer
of epidermal cells with a layer of hypodermal cells.
152 Int. Res. J. Plant Sci.
a
b
e
f
c
d
g
h
Plate 1. Photomicrographs of epidermal layers of leaves of Gardenia erubescens and Gardenia ternifolia
Key: a and b – adaxial surfaces of G. erubescens; c and d – abaxial surfaces of G. erubscens; e and f – adaxial surfaces of
G. ternifolia; g and h – abaxial surfaces of G. ternifolia; ep – epidermal cells; hy – hypodermal cells; sc – Subsidiary cells; cr –
Crystals; st – stomata
st
cr
Plate 2. Photomicrographs of Abaxial Epidermal layers of leaves of Gardenia erubescens and Gardenia
ternifolia
Key: a: Gardenia erubescens; b: Gardenia ternifolia. cr – crystals; st – stomata. Mag. X40
Hypodermal cells not continuous as in G. erubescens but
intermingled with palisade cells (Plates 3D and E). Two
layers of palisade cells; Idioblast cells with rosette
crystals not as abundant as in G. erubescens and also
restricted in between hypodermal cells (Plates 3D and E).
Oil globules on palisade, mesophyll and lower epidermal
cells. Fibers abundant, short xylem vessels and
isodiametric sclereids (Plates 3D and E). Presence of
sparse non-glandular multicellular trichomes in between
the midrib and laminar (Plates 3G and H) table 1
Ibrahim et al. 153
ep
cr
pal
ep
fb
vb
ep
hy
hy
sc
ep
pal
tr
hy
vb
tr
Plate 3. Photomicrographs of Transverse sections of leaves of Gardenia erubescens and Gardenia ternifolia
Key: A – transverse section of Gardenia erubescens Mag. X100; B – transverse section of G. erubescens Mag. X400; C –
midrib of G. erubescens Mag. X100; D – transverse section of G. ternifolia Mag. X100; E – transverse section of G.
ternifolia Mag. X400; F – midrib of G. ternifolia Mag. X100; G and H – showing trichomes of G. ternifolia; ep – epidermal
cells; hy – hypodermal cells; cr – crystals; fb – fiber; pal – palisade cells; vb – vascular bundle; sc – sclereids; tr trichomes
Table 1. Result of the Physicochemical parameters of the leaves of Gardenia erubescens and Gardenia ternifolia
Parameter
Moisture content
Total ash value
Acid – insoluble ash value
Alcohol – extractive value
Water – extractive value
Gardenia erubescens (% w/w)
9.1
5.6
2.8
36.4
19.0
Gardenia ternifolia (%w/w)
10.7
5.3
2.4
22.4
18.8
154 Int. Res. J. Plant Sci.
Table 2. Result of the Phytochemical analysis of the leaves of Gardenia erubescens and
Gardenia ternifolia
Secondary Metabolite
Carbohydrate
Tannins
Saponins
Flavonoids
Anthraquinones
Terpenes
Balsams
Resins
Alkaloids
Cardiac Glycoside
Sterols
Phenols
Gardenia
erubescens
+
+
+
+
+
+
+
+
+
+
+
Gardenia
ternifolia
+
+
+
+
+
+
+
+
+
+
+
Key:
+ = positive
- = negative
Table 3. Nature of the leaf extract of Gardenia erubescens and Gardenia ternifolia from
successive extraction
Gardenia erubescens
Gardenia ternifolia
n- Hexane extract
Liquid
Liquid
Thin layer chromatography (TLC) of the extracts
The hexane and ethyl acetate extracts gave 4 spots each
for Gardenia erubescens while for G. ternifolia 4 and 5
spots respectively were obtained (Table 4; Plates 4a, b, e
and f). After spraying the plates with sulphuric acid, G.
erubescens gave 6 and 7 spots for hexane and
ethylacetate extracts respectively while G. ternifolia gave
7 spots each for both extracts (Table 4; Plate 4h, i, k and
l). A different solvent system was used for the methanol
extract and gave 2 spots each before it was sprayed with
sulphuric acid (Table 4; Plate5a andb). After spraying, 5
spots were obtained for G. erubescens while G. ternifolia
showed 4 spots (Table 5; Plates 3c and d).
DISCUSSION
The result of the phytochemical screening revealed
presence of similar metabolites in the two species. The
metabolites present were tannins, saponins, flavonoid,
anthraquinone, terpenes, balsam, alkaloid, cardiac
glycoside, sterols and phenols. The usefulness of these
metabolites in phytomedicines or treatment of ailments
has been documented. Tannins have anti-diarrhoeal
activity and also used for treatment of sexually
Ethylacetate extract
Liquid
Liquid
Methanol extract
Jelly (semi-solid)
Liquid
transmitted diseases; saponins are used for gastrointestinal infection; Flavonoids are free radical
scavengers and therefore useful in management of
inflammatory diseases e.g tumour and oxidative stress –
related diseases (Robertson and Heber, 1956; Haslem,
1989; Evans, 2002). Anthraquinones can be used as
laxatives, terpenes have anti-cancer and anti-malaria
activities, phenols have antiseptic property (Robertson
and Heber, 1956; Haslem, 1989; Evans, 2002).
Therefore, the presence of these metabolites in these
plants supports their uses in treatment of various
ailments traditionally (Burkil, 1997).
Extracts obtained from successive extraction of
leaves of both species using N-hexane, ethylacetate and
methanol were liquid in nature but the methanol extract of
G. erubescens was semi-solid or jelly-like in nature which
is a distinguishing feature for G. erubescens. The TLC
fingerprinting confirmed the presence of secondary
metabolites on the plants and can be used to identify,
standardize and differentiate the two species. This is
really useful especially in cases of adulteration.
Epidermal characteristics observed can also be used
to differentiate the two species. For example, view of
epidermal cells and large irregular shaped hypodermal
cells and abundant oil globules on the adaxial surface;
and abundant cyclic stomata type, polygonal cells and
Ibrahim et al. 155
Table 4. Retardation factors (Rf) and colour of components of the extracts of the leaves of Gardenia erubescens and Gardenia ternifolia
No. of
spots
1.
2.
3.
4.
5.
6.
7.
Hexane extract
BS
AS
0.92
0.9 (dark
(yellow)
brown)
0.50
0.50 (ash)
(green)
0.39 (light
0.51
green)
(green)
0.17 (light
0.44
yellow)
(orange)
0.38 (light
green)
0.28
(purple)
Gardenia erubescens
Ethylacetate extract
BS
AS
0.72 (faint
0.94 (pink)
green)
0.50 (green)
0.72 (light
green)
0.39 (light
0.51
green)
(green)
0.17 (yellow)
0.44
(orange)
0.38 (light
green)
Methanol extract
BS
AS
0.95 (light
0.95
green)
(green)
0.80 (light
0.91
green)
(green)
0.86
(brown)
0.80 (light
green)
0.62
(yellow)
Hexane extract
BS
AS
0.92
0.88
(yellow)
(brown)
0.53
0.78 (ash)
(green)
0.34 (light
0.57 (light
green)
green)
0.17
0.47
(yellow)
(green)
0.34 (light
green)
0.33
(brown)
0.28
(purple)
0.29 (pink)
0.17
(brown)
KEY: BS: Before spraying with sulphuric acid; AS: After spraying with sulphuric acid
After spraying
Before spraying
a
b c
e
f
g
h i
j
k
l
m
Plate 4. Chromatogram of extracts of Gardenia erubescens and
Gardenia ternifolia before and after spraying with dilute sulphuric acid
Key: G.e: Plate of Gardenia erubescens; G.t: Plate of Gardenia ternifolia;
a: spot of hexane extract; b: spot of ethylacetate extract; c: spot of
methanol extract
Gardenia ternifolia
Ethylacetate extract
BS
AS
0.75 (faint
0.75
green)
(green)
0.53
0.69
(green)
(pink)
0.34 (light
0.52
green)
(green)
0.17
0.44
(yellow)
(pink)
0.08
0.37
(green)
(light
green)
0.29
(pink)
0.17
(brown)
Methanol extract
BS
AS
0.95 (light
0.95
green)
(green)
0.77
0.91 (pink)
(green)
0.80
(brown)
0.74 (light
green)
156 Int. Res. J. Plant Sci.
Before spraying
a
b
After spraying
c
d
Plate 5. Chromatogram of methanolic extract of Gardenia
erubescens and Gardenia ternifolia before and after spraying with
dilute sulphuric acid
Key:G.e: Spot of Gardenia erubescens; G.t: spot of Gardenia
ternifolia;
abundant crystals on the abaxial surface of G.
erubescens are diagnostic features peculiar to the
species. The characteristic and diagnostic features of G.
ternifolia are the absence of hypodermal cells on adaxial
surface, abundant anomocytic stomata type, irregular
shaped epidermal cells and abundant oil globules on the
abaxial surface (Plate 1).
The transverse sections of the leaves also revealed
diagnostic characters which can be used to distinguish
the two species even when only leaf fragments are
available. The hypodermal cells of G. erubescens were
continuous and had three layers of palisade cells while
those of G. ternifolia were not continuous but
intermittently mixed with palisade cells and there were
only two layers of palisade cells. The presence of
isodiametric sclerieds and non-glandular multicellular
trichomes in between the midrib and lamina of G.
ternifolia were also pecuiar to this species. The presence
of sclereids in the leaves of G. ternifolia might account for
the leathery nature of the leaves of this species.
In the qualitative evaluation of the powdered leaf
samples, the low value of 5.6%w/w and 2.8%w/w of total
ash value and acid – insoluble ash value respectively for
G. erubescns and total ash value of 5.3%w/w and
2.4%w/w of acid – insoluble ash value for G. ternifolia
indicates that the plant contain a lot of organic than
inorganic compounds, indicating the usefulness of the
plant. The alcohol extractive values were greater than the
water extractive values, indicative that alcohol would be a
better solvent for extraction. The values of the moisture
content (9.1%w/w and 10.7%w/w) were between the
official range of 8 – 14% for vegetable drugs (African
pharmacopeia, 1989). This implied that both plant
samples had a low chance for microbial attack when
stored under good condition.
CONCLUSION
The two species of Gardenia evaluated were rich in
secondary metabolites and therefore could be potential
sources of phytomedicines and leads for synthetic drugs.
Further studies are on-going to verify the claimed
traditional uses of these plants. The diagnostic characters
generated from this study would be useful in identification
of these plants especially when the leaves are in
fragments. The result would also be useful in
standardization of the plants towards quality assurance
and in preparation of monograph on the plants.
ACKNOWLEDGMENT
The authors wish to acknowledge the contribution of
Mall. Muazzam Ibrahim who help in the collection of the
plants.
Ibrahim et al. 157
REFERENCES
Africa Pharmacopeia (1986). General Method of Analysis. (OAU/STRC)
Lagos. 2:126-150
Ainslie JR (1937). A list of Plants used in Native Medicine in Nigeria.
Imp. Forest. Inst. Oxford Inst. Paper. sp no. 165
Akinnbiyi JA, Sultanbawa MUS (1983). A glossary of Kanuri names,
with botanical names, distribution and uses. Ann. Borno. 1, University
of Maiduguri.
Burkil HM (1997). The useful plants of West Tropical Africa. 2nd ed.
Families M – R. Royal Botanic Gardens. Kew,4:538-544.
El Ghazali GEB, Bari EA, Bashir AK, Salih AKM (1987). Gardenia
ternifolia. In: Medicinal Plants of the Sudan: Part 11, Medicinal Plants
of the Eastern Nuba Mountains. Khartoum University Press.: 98-99.
Evans WC (1996). Trease and Evans Pharmacognosy. 14th edition.
W.B. Saunder Company Ltd. London; 105-108; 545-578
Evans WC (2002). Trease and Evans Pharmacognosy, 15th Ed. London:
W.B. Sanders. 183-393.
Farah HM, El Amin TH, Khalid HS, Hassan SM, El Hussein ARM
(2012). In vitro activity of the aqueous extract of Gardenia ternifolia
fruits against Theileria lestoquardi. Journal of Medicinal Plants
Research. 6(41): 5447-5451
Haslem E (1989). Plant polyphenols: Vegetable tannins revisited –
chemistry and pharmacology of natural products. Cambridge
University Press, Cambridge, pp. 169.
Irvine FR. (1961). Woody plants of Ghana. Oxford University Press,
London, 585-586
Kerharo J, Adam JG (1962). Premier Inventaire des Plantes
Medicinales et toxiques de la Casamance (Senegal). Ann. Pharm.
Franc. 20: 726-744, 823-841
Kerharo J, Adam JG (1964). Plantes Medicinales et toxiques des Peul
et des Toucoulour du Senegal. J. Agri. Trop. Bot. Appl.; 11: 419
Kerharo J, Bouquet A (1950). Plantes medicinales et toxiques de la
Cote d’Ivoire-Haute Volta. Vigot Freres, Paris., \179.
Robertson P, Herber WY (1956). Pharmacognosy. J.B Lippincott
Company; 123-125.
Sofowora A (2008). Medicinal Plants and Traditional Medicine in Africa.
3rd Ed. Spectrum Books Limited, Ibadan, Nigeria; 199-204.
Walker AR (1953). Usages Pharmaceutiques des Plantes Spontaneous
du Gabon, 11. Bull. Inst. Etudes Centrafr. n.s 5:19-40