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CHAPTER 2

PHARMACOGNOSY: MORPHOLOGICAL, ANATOMICAL AND

PROXIMATE ANALYSIS OF LEAF, ROOT AND RHIZOME OF

COSTUS IGNEUS (N.E.Br.)

2.1 INTRODUCTION

The Costaceae was first raised to the rank of family by Nakai (1941). The

family is one of the most distinctive and isolated members of the order Zingiberaceae

(Dahlgren et al., 1985). Before the elevation to family status, Engler and Prantl

(1930) recognized Costoideal as a subfamily under Zingiberaceae. Several anatomical

and morphological features support this isolated position including well developed

arial shoots with distinct, rigid and commonly branched stems. The leaves are

inserted in a low spiral with divergences.

The family Costaceae consists of four genera and approximately 200 species

(Airy Shaw, 1973). The genus Costus is the largest in the family with about 150

species that are mainly tropical in distribution. In addition to its small size and

distinctive morphology, Costus has a pan tropical distribution especially in the forest

and savanna regions. Tomlinson (1956) presented some reasons for treating the

Costaceae as a separate family based on a number of anatomical characters of the

vegetative organs. Some of these diagnostic characters were summarized by

Tomlinson (1956). These include the uniseriate filamentous, hairs isodiametric,

irregular, or transversely extended thin walled epidermal cells, tetracytic stomata

(stomata with subsidiary cells), slightly asymmetrical nature of guard cells, colours

and continuous hypodermis, clearly differentiated cortex, well developed vascular

bundle at each node restriction of silica to internal tissue close to vascular bundles,

solitary bodies stellately spherical or drug like vessels restricted for root, etc.

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(Edeoga, 1991). Apart from preliminary investigation of Tomlinson (1956) on the

anatomy of some members of Costaceae no other information of costus igneus leaf,

rhizome and root anatomy have been documented to the best of our knowledge.

2.2 MATERIALS AND METHODS

2.2.1 Collection of plant material

Costus igneus were grown at Periyar Maniammai University nursery and were

collected during the month of April (2008).The identification of the plant was

confirmed and authenticated by Rapinant Herbarium, St. Joseph’s College, Trichy,

Tamilnadu, South India. Freshly collected samples were washed and used for study of

organoleptic and microscopic characteristics. The dried powder samples were used

for further analysis. All chemicals and reagents used in this study were analytical

grade obtained from Hi Media, Qualigens and Loba Chemicals, available in India.

2.2.2 Morphological and Anatomical studies of Costus igneus

2.2.2.1 Specimens preparation

Leaf, root and rhizome of Costus igneus obtained from living specimen of

plant were fixed in FAE (1:1:18) (formalin – 5ml + Acetic acid -5ml + 70% ethyl

alcohol-90 ml) for 48-72 h. The specimens were dehydrated with graded series of

tertiary butyl alcohol (TBA) as per the schedule given by Sass (1940). Infiltration of

the specimens was carried by gradual addition of paraffin wax (melting point 58°-

60°C) until TBA solution attains super saturation. The specimens were cast into

paraffin blocks.

2.2.2.2 Sectioning

The paraffin embedded specimens were sectioned with the help of Rotary

Microtome. The thickness of the sections was 10-12 µm. Dewaxing of the section

was by customary procedure (Johansen, 1940). The sections were stained with

Toluidine Blue as per the method published by O’Brien et al., (1964). The dye

rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to

23

submarine, violet to the mucilage, blue to the protein bodies, etc., Wherever

necessary sections were also stained with safranin and fast-green. For studying the

stomatal morphology, venation pattern and trichome distribution, paradermal sections

(sections taken parallel to the surface of leaf) as well as clearing of leaf with 5%

sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey’s

maceration fluid (Sass, 1940) were prepared. Glycerine mounted temporary

preparations were made for macerated/cleared materials. Powdered materials of

different parts were cleared with NaOH and then mounted in glycerine medium after

staining. Different cell components were studied and measured.

2.2.2.3 Photomicrography

Photographs of different magnifications were taken with Nikon lab photo 2

microscopic units. For normal observations bright field was used. For the study of

crystals, starch grains and lignified cells polarized light was employed. Since these

structures have birefringent property, under polarized light they appear bright against

dark background. Magnification of the figures is indicated by the scale-bars.

Descriptive terms of the anatomical features are as given in the standard plant

anatomy text books.

2.2.3 Proximate Analysis

The proximate analysis namely total ash content, acid insoluble ash, water

soluble ash and moisture content were determined by the methods described by

A.O.A.C (1990). The total carbohydrate and total protein were also determined by

Anthrone method (Hedge and Hofreiter, 1962) and Lowry method (Lowery et al.,

1953) respectively.

2.2.4 Statistical analysis

The proximate analysis was carried out in triplicate for four separate

experiments. The results were expressed as Mean ± Standard Deviation.

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2.3 RESULTS

2.3.1 Morphological characters

The Costus igneus is a rhizomatous shrub and penetrates through the tuberous

rhizome. The rhizome is about 20-40cm grown, cylindrical, soft and fleshly with

smooth pale brown surface. It is pleasantly aromatic. The leaves are green in color.

Their length are about 15-25cm and narrow with several parallel equally thick veins.

Tap root sub-cylindrical, wider at the top 28-50cm long, outer surface light brownish

to pale dark brown (Figs. 2.1.1-2.1.3).

2.3.2 Anatomical studies of Costus igneus

2.3.2.1Anatomy of Rhizome

The rhizome is circular with smooth and even surface. It has fairly distinct

layer of epidermis, which consist of narrow oblong thin walled cells. Inner to the

cortex is a wide cortical zone in radial plane. The cortical cells are circular wavy, thin

walled, compact and possess wide spread structure. Randomly distributed in the

cortex are small circular cortical vascular bundles (Figs. 2.3.1 and 2.3.2). The cortical

bundles are collateral with a wide, angular fairly thick walled xylem elements and

small cluster of phloem elements (Figs. 2.3.2 and 2. 4.1). Prominent clusters of xylem

elements occur associated with the endodermoid layer (Fig. 2.3.2). The xylem

elements are in tangential multiples; they are wide, angular, thick walled and measure

50µm in diameter. Phloem elements also occur as a thin sheath in-between the

endodermis and xylem elements. The stele has an outer boundary of endodermoid

layer, which is not well defined (Fig. 2.2.2). The stelar bundles are more numerous

and scattered in the stele. They are also collateral and they are different in orientation

(Fig. 2.4.2). The central (stelar) vascular bundles have wide, angular, thin walled

metaxylem elements and three or four proto xylem elements (Fig. 2.4.2). The

metaxylem elements are 70µm wide. Thick mass of phloem occurs adjacent to the

meta xylem elements. The phloem elements are wide and prominent.

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2.3.2.2 Anatomy of Leaf

The leaf is thin with smooth even surfaces, isobilateral and has no difference

between the upper and lower sides. The leaf has two layers of thin epidermal cells and

four layers of wide, tangentially oblong thin walled mesophyll cells. Both epidermal

layers have narrow, tangentially flat thin walled cells (Fig. 2.3.1); they are 10-20µm

thick. The mesophyll cells are 100-140µm thick. These are prominent vascular

bundles placed in the median part of the lamina. The bundles are collateral having

wide mass of xylem elements and a small cluster of phloem. On the phloem end of

the bundle, there is thick band of sclerenchyma cells are lignified (Figs. 2.3.2 and

2.3.3). The xylem elements are wide, angular and thick walled. The vascular bundles

have no distinct bundle sheath cells.

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Fig. 2.1.1 Costus igneus leaf

Fig. 2.1.2 Costus igneus rhizome Fig. 2.1.3 Costus igneus root

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Fig. 2.2.1 Rhizome outer sector (CB-Cortical vascular bundle; CO- Cortex)

Fig. 2.2.2 Rhizome inner sector (EV-Endodermoid vascular bundle; SB-Stelar

bundle;St- stele)

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Fig. 2.2.3 Cortex with outer cortical bundle (CO-Cortex; Ep-Epidermis; OCB-

outer cortical bundle)

Fig. 2.2.4 Inner cortical bundle with stelar vascular bundle (CO-Cortex; ICB-Inner

cortical Bundle; OC-Oil contents; En-Endodermis; Ph- Phloem; St-Stele; X-

Xylem Ph- Phloem)

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Fig. 2.2.5 Endodermoid Vascular Bundle Magnified (CO-Cortex; End-

Endodermoid layer; Ph- Phloem; X- Xylem)

Fig. 2.2.6 Central stelar Vascular Bundle Magnified (GP-Ground parenchyma;

SG- Starch grains; OC- Oil content; Ph- Phloem; X-Xylem)

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Fig. 2.2.7 Cortical vascular bundle enlarged (MX- Metaxylem; Ph- Phloem)

Fig. 2.2.8 Stelar vascular bundle enlarged (MX- Metaxylem; Ph- Phloem; PX-

Proto xylem; SE- Sieve elements)

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Fig. 2.3.1 T.S of leaf through lamina with vascular bundle (AbE- Abaxial

epidermis; AdE- Adaxial epidermis; MT- Mesophyll tissue; VB- Vascular bundle)

Fig. 2.3.2 Vascular bundle of the leaf enlarged (Ph- Phloem; SC- Sclerenchyma;

X- Xylem)

Fig. 2.3.3 Starch grains in the rhizome (under polarized light microscope) (SG-

Starch grains)

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Fig. 2.4.1 T. S of root a sector (CO- Cortex; X- Xylem; SPe- Stored periderm)

Fig. 2.4.2 Same as above a portion enlarged (CO- Cortex; En- Endodermis; MX-

Metaxylem; PC-Pericycle; Pi- Pith; Px-Protoxylem; SC-Sclerenchyma; SPe-

Stored periderm; X-Xylem)

Fig. 2.4.3 One vascular bundle magnified (MX- Metaxylem; En- Endodermis;

PC-Pericycle; Px- Protoxylem;SC- Sclerenchyma; Ph- Phloem)

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2.3.2.3 Anatomy of Root

Thin roots have fairly wide superficial sequent periderm and narrow

homogenous paranchymatous cortex. In olden thick root, the periderm is slightly

wider and consists of polyhedral, randomly oriented, thin walled cells. The cortex is

wider and parenchymatous with small, thin walled compact cells (Fig. 2.4.2). The

periderm is 150µm wide and the cortex is 400µm wide. The stele has about 10 exarch

xylem alternating with equal numbers of phloem strands (Fig. 2.4.1). The stele has

distinct endodermis with cylindrical narrow cells; their inner and lateral walls have

U-shaped thickenings. Inner to the endodermis is a narrow layer of hyaline cells,

which are circular to rectangular in outline. The xylem strands are numerous and each

strand has a wide circular, thick walled metaxylem elements and narrow proto xylem

elements; the metaxylem is 60-70µm wide (Fig. 2.4.3); the proto xylem elements are

40 µm wide. A prominent mass of phloem occurs in between the xylem. No

sclerenchyma tissue is seen in the stele.

2.3.3 Proximate analysis

The result of fluorescent analysis, ash (total, acid soluble, water soluble)

content, moister content, carbohydrate and protein of Costus igneus leaf, root and

stem are represented in Tables 2.1, 2.2 and 2.3 respectively.

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Table 2.1 Fluorescent analysis of leaf, stem and rhizome powder of Costus igneus

under UV light and daylight

S.No Chemical

Test

Leaf Rhizome Stem

Day light UV light Day light UV light Day light UV light

1 Sample as

such

Yellowish

brown

Light

green Brown

Greenish

brown

Light

brown Green

2

Extract

with

aqueous

NaOH

Dark

brown

Light

brown

Dark

brown Green

Dark

Brown Brown

3

With

alcohol

NaOH

Brown Light

green

Dark

brown Green Brown

Light

green

4 With HCl Yellowish

brown

Light

green

Light

brown

Light

green

Light

brown

Light

green

5 With 50%

HNO3

Dark

brown

Dark

green

Light

brown

Dark

green Brown Green

6 With 50%

H2SO4

Reddish

brown

Blackish

green

Reddish

brown

Dark

green

Light

reddish

brown

Blackish

green

7 Methanol Yellowish

black

Yellowish

black

Dark

green

Light

brown

Yellowish

brown Brown

8 With

ammonia

Light

brown

Light

green

Yellowish

brown

Light

green

Yellowish

brown Brown

9 With I2

solution

Blue

Brownish

Blackish

green

Blue

Brownish

Blackish

green

Blue

Brownish Black

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Table 2.2 Behavior of leaf, rhizome and stem powder of Costus igneus with

different chemical reagents

Reagent Sample

Name Colour / ppt Constituent

Conc.Sulphuric

acid

Leaf Reddish brown Steroids

Rhizome Reddish brown Steroids

Stem Light reddish brown Steroids

Aqueous Ferric

chloride solution

Leaf Dark Blackish brown Tannin

Rhizome Blackish brown Tannin

Stem Blackish brown Tannin

Iodine solution

Leaf Blue’s brown Starch

Rhizome Blue’s brown Starch

Stem Blue’s brown Starch

Ammonia

solution

Leaf Dark yellowish brown Anthraquinone

Rhizome Yellowish brown Anthraquinone

Stem Light Yellowish brown Anthraquinone

Aqueous

Potassium

hydroxide

Solution (5%)

Leaf Yellowish brown Anthraquinone

Rhizome Yellowish brown Anthraquinone

Stem Light Yellowish brown Anthraquinone

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Table 2.3 Proximate analysis of rhizome, stem and leaf of Costus igneus

S.NO Parameters Costus igneus

Root Stem Leaf

1 Total ash (%) 4.20±0.007c 2.20±0.026

2.20±0.026

2 Acid insoluble (%) 0.20±0.020 0.13±0.073 0.17±0.022

3 Water soluble (%) 0.74±0.0267 1.00±0.027 1.00±0.027

4 Moisture (%)

(After 1 hour) 5.40±0.040

b 10.40±0.187

14.00±0.427

5 Moisture (%)

(After 1.5 hour) 7.40±0.393 14.36±0.063 21.20±0.447

6 Carbohydrate (g/kg) 9.31±0.007a 7.87±0.016

b, 11.20±0.107

7 Protein (g/kg) 2.78±0.002a 1.60±0.016

a, , 3.30±0.06

The results were expressed as mean ± Standard deviation; Statistical significance variation

were compared leaf Vs stem, rhizome; at ap<0.05,

bp<0.01,

cp<0.001,

d p< 0.0001; rhizome

Vs stem at,

p<0.05,

p<0.01, ,

p<0.001.

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2.4 DISCUSSION

The Costaceae plants were identified to have different morphological and

anatomical characters of leaf, rhizome and root among them Costus igneus have a

characteristic anatomy that could be used to distinguish it from other members of

Costaceae. Costus igneus studied, however, appear to be a homogenous entity united

by series of leaf, rhizome and root anatomical characters, including epidermal cells,

mesophyll cells, the sclerenchyma cells, vascular bundle, cortex, xylem, phloem,

starch grains, stelar bundle. These features of lamina, rhizome and root anatomy

endorse. The finding of Oteng-Yeboah (1981) in the same species of Costus from

West Africa coincides with our present study. Differences in vegetative anatomy

among such members of Costaceae as Costus and Tapeinochilus (Tomlinson, 1956)

have received some emphasis in phylogenetic discussion. The anatomical structures

of leaf, rhizome and root of Costus igneus differentiated that of C. lucanusianus and

C. afer that was studied by Edeoga and Okoli (1997). The leaf has two layers of thin

epidermal cells and four layers of wide, tangentially oblong thin walled mesophyll

cells. It is 300µm thick. Randomly distributed in the cortex are small circular cortical

vascular bundles. The cortical bundles are collateral with a wide, angular fairly thick

walled xylem elements and small cluster of phloem elements. Thin roots have fairly

wide superficial sequent periderm and narrow homogenous paranchymatous cortex.

The stele has about 10 exarch xylem alternating with equal number of phloem

strands. No sclerenchyma tissue is seen in the stele. Tomlinson (1956) presented

some reasons for treating Costaceae as a separate family from Zingiberaceae. The

lamina, rhizome and root anatomy of these Costus species investigated seems to agree

with the Tomlinson’s (1956) reasons for supporting the raising of the genus to rank of

family by Nakai (1941). Cytology, phytochemistry of Costus igneus studies helps to

clearly differentiate Costus igneus from other Costaceae plant. Tomlinson (1956)

outlined some diagnostic features of the leaf, stem, rhizome and root anatomy in the

Zingiberales as a whole but did specify how these could be used in resolving critical

systematic problem as in the presently investigated Costus igneus anatomical study.

The proximate values shows that the protein content is relatively low but it can

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contribute to the formation of hormones which controls a variety of body functions

such as growth, repair and maintenance of body protein (Mau et al., 1999). The

relatively high carbohydrate content can be used as energy source and also it is

necessary in the digestion and assimilation of other foods. The moisture and ash

content is useful in assessing the quality of grading the plant and also gives an idea of

the amount of minerals present in the samples (Michael and David, 2002). At present

investigation, Costus igneus have a characteristic anatomy and proximate analysis

that could be used to distinguish it from other members of Costaceae.