Journal of Pharmacy Research Vol.3.Issue 4.April 2010

K. Pazhanichamy. et al. / Journal of Pharmacy Research 2010, 3(4),747-752

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Research ArticleISSN: 0974-6943 Available online through

www.jpronline.info

*Corresponding author.Dr.T.EeveraDepartment of Biotechnology, Periyar Maniammai University, Vallam-613403,Tamilnadu, India.Tel.: + 91-09790035761Telefax: +91-04362-264660E-mail:teevera2000@yahoo.com

Morphological, anatomical and proximate analysis of Leaf, Root, Rhizome of Costus igneusK. Pazhanichamy., S. Pavithra., S. Rubini., B. Lavanya., I. Ramya and T. Eevera.*Department of Biotechnology, Periyar Maniammai University, Vallam-613403, Tamilnadu, IndiaReceived on: 12-11-2009; Revised on: 29-12-2009; Accepted on:18-02-2010

ABSTRACT

This study is based on anatomical and morphological investigations of Costus igneus. It is an important plant because of its value for medicinal uses. Plantsamples were cultivated in Periyar Maniammai University nursery under net house and the morphological, anatomical features of various plant parts ofCostus igneus such as young root, leaf and rhizome were investigated in detail and demonstrated by illustrations. The total ash value, acid insoluble ash andwater-soluble ash of rhizome were significantly (p<0.001) higher than stem and leaf. Moisture content, protein and carbohydrate were significantly higherin leaf. In Fluorescent analysis, leaf, stem and rhizome powder were treated with various chemicals and were studied under UV light and daylight. At presentfinding, Costus igneus have characteristic morphological, anatomical and proximate features that could be used to distinguish it from other members of theCostaceae family.

Keywords: Morphology, Anatomy, proximate analysis, Costus igneus, Leaf, Rhizome

INTRODUCTION

The Costaceae was first raised to the rank of family by Nakai(1941). The family is one of the most distinctive and isolated membersof the order Zingiberaceae (Dahlgren et al., 1985). Before the eleva-tion to family status, Engler and prantl (1930) recognized Costoidealas a subfamily under Zingiberaceae. Several anatomical and morpho-logical features support this isolated position including well devel-oped 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 approxi-mately 200 species (Airy Shaw, 1973). The genus Costus is the largestin the family with about 150 species that are mainly tropical in distri-bution (Humphries 1985 and Hickey 1981). In addition to its small sizeand distinctive morphology, Costus has a pantropical distributionespecially in the forest and savanna regions. Tomlinson (1956) pre-sented some reasons for treating the Costaceae as a separate familybased 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, tetracyticstomata (stomata with subsidiary cells), slightly asymmetrical natureof guard cells, colours and continuous hypodermis, clearly differenti-ated cortex, well developed vascular bundle at each nod restriction ofsilica to internal tissue close to vascular bundles, solitary bodiesstellately spherical or drug like vessels restricted for root etc (Edeoga,1991). Apart from preliminary investigation of Tomlinson (1956) onthe anatomy of some members of Costaceae no other information on

costus igneus leaf, rhizome and root anatomy have been documentedto the best of our knowledge.

MATERIALS AND METHODS

The fresh leaves, rhizome and root of Costus igneus werecollected From Periyar Maniammai University nursery in the month ofApril (2008). These were identified, confirmed and authenticated byRapinant Herbarium, St. Joseph’s College, Trichy, Tamilnadu, SouthIndia. Collected fresh samples were washed and used for study oforganoleptic and microscopic characteristics. The powder of driedsamples was used for further analysis. All chemicals and reagentsused for testing were analytical grade obtained from Hi media,Qualigens and Loba Chemicals.

Collection of Specimens

Leaf, root and rhizome of Costus igneus obtained from livingspecimen of plant were fixed in FAE (1:1:18) (formalin – 5ml + Aceticacid -5ml + 70% ethyl alcohol-90 ml) for 48-72 hours. The specimenswere dehydrated with graded series of tertiary butyl alcohol (TBA) asper the schedule given by Sass (1940). Infiltration of the specimenswas carried by gradual addition of paraffin wax (melting point 58°-60°C) until TBA solution attains super saturation. The specimenswere cast into paraffin blocks.

Sectioning

The paraffin embedded specimens were sectioned with thehelp of Rotary Microtome. The thickness of the sections was 10-12µm. Dewaxing of the section was by customary procedure (Johansen,1940). The sections were stained with Toluidine Blue as per the method

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published by O’Brien et al., (1964). The dye rendered pink colour tothe cellulose walls, blue to the lignified cells, dark green to submarine,violet to the mucilage, blue to the protein bodies etc., Wherever nec-essary sections were also stained with safranin and fast-green. Forstudying the stomatal morphology, venation pattern and trichomedistribution, paradermal sections (sections taken parallel to the sur-face of leaf) as well as clearing of leaf with 5% sodium hydroxide orepidermal peeling by partial maceration employing Jeffrey’s macera-tion fluid (Sass, 1940) were prepared. Glycerine mounted temporarypreparations were made for macerated/cleared materials. Powderedmaterials of different parts were cleared with NaOH and then mountedin glycerine medium after staining. Different cell components werestudied and measured.

Photomicrography

Photographs of different magnifications were taken withNikon lab photo 2 microscopic units. For normal observations brightfield was used. For the study of crystals, starch grains and lignifiedcells polarised light was employed. Since these structures have bire-fringent property, under polarised light they appear bright againstdark background. Magnification of the figures is indicated by thescale-bars. Descriptive terms of the anatomical features are as givenin the standard anatomy books (Esau, 1964).

Proximate Analysis

The proximate analysis namely total ash content, acid in-soluble ash, water soluble ash and moister content were determinedby the methods described by A.O.A.C (1990). The total carbohydrateand total protein were also determined by Anthrone method (HedgeJ. E and Hofreiter, B.T, 1962) and Lowry method (lowery, O.H et al.,1953) respectively.

Statistical analysis

The proximate analysis was carried out in triplicate for fourseparate experiments. The results were expressed as mean ± Standarddeviation.

RESULTS

Macroscopic characters

The plant is a rhizomatous shrub and penetrates throughthe tuberous rhizome. The rhizome is about 30-40cm In length, cylin-drical, soft and fleshly with smooth pale brown surface. It is pleasantlyaromatic. The leaves are green in color. Their length are about 18-

25cm and narrow with several parallel equally thick veins. Tap rootsub-cylindrical, wider at the top 28-50cm long, outer surface lightbrownish to pale dark brown (Fig. 1A-C).

Anatomy of Rhizome

The rhizome is circular with smooth and even surface. It hasfairly distinct layer of epidermis, which consist of narrow oblong thinwalled cells. Inner to the cortex is a wide cortical zone in radial plane.The cortical cells are circular wavy, thin walled, compact and possesswide spread structure. Randomly distributed in the cortex are smallcircular cortical vascular bundles (Fig. 3A-B). The cortical bundlesare collateral with a wide, angular fairly thick walled xylem elementsand small cluster of phloem elements (Fig. 3B and 4A). Prominentclusters of xylem elements were associated with the endodermoidlayer (Fig. 3B).The xylem elements are in tangential multiples; theyare wide, angular, thick walled and measure 50µm in diameter. Phloemelements also occur as a thin sheath in-between the endodermis andxylem elements. The stele has an outer boundary of endodermoidlayer, which is not well defined (Fig. 2B). The stelar bundles are morenumerous and scattered in the stele. They are also collateral and theyare different in orientation (Fig. 4B). The central (stelar) vascularbundles have wide, angular, thin walled metaxylem elements and threeor four proto xylem elements (Fig. 5B). The metaxylem elements are 70µm wide. Thick mass of phloem occurs adjacent to the meta xylemelements. The phloem elements are wide and prominent.

Anatomy of Leaf

The leaf is thin with smooth even surfaces, isobilateral andhas no difference between the upper and lower sides. The leaf hastwo layers of thin epidermal cells and four layers of wide, tangentiallyoblong thin walled mesophyll cells. Both epidermal layers have nar-row, tangentially flat thin walled cells (Fig. 6A); they are 10-20 µmthick. The mesophyll cells are 100-140 µm thick. These are prominentvascular bundles placed in the median part of the lamina. The bundlesare collateral having wide mass of xylem elements and a small clusterof phloem. On the phloem end of the bundle, there is thick band ofsclerenchyma cells are lignified (Fig. 6B). The xylem elements arewide, angular and thick walled. The vascular bundles have no dis-tinct bundle sheath cells.

Anatomy of Root

Thin roots have fairly wide superficial sequent periderm andnarrow homogenous paranchymatous cortex. In olden thick root, theperiderm is slightly wider and consists of polyhedral, randomly ori-ented, thin walled cells. The cortex is wider and parenchymatous with

Table 1 Fluorescent analysis of Leaf and Root powder of Costus igneus under UV light and daylight

S.No Chemical Test Leaf Root Stem

Day light UV light Day light UV light Day light UV light1 Sample as such Yellowish brown Light green Brown Greenish brown Light brown Green2 Extract with aq. NaOH Dark brown Light brown Dark brown Green Dark Brown Brown3 With alc. NaOH Brown Light green Dark brown Green Brown Light green4 With HCl Yellowish brown Light green Light brown Light green Light brown Light green5 With 50% HNO3 Dark brown Dark green Light brown Dark green Brown Green6 With 50% H 2SO4 Reddish brown Blackish green Reddish brown Dark green Light reddish brown Blackish green7 Methanol Yellowish black Yellowish black Dark green Light brown Yellowish brown Brown8 With ammonia Light brown Light green Yellowish brown Light green Yellowish brown Brown9 With I2 solution Blue Brownish Blackish green Blue Brownish Blackish green Blue Brownish Black

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Table 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 SteroidsRhizome Reddish brown SteroidsStem Light reddish brown Steroids

Aqueous Ferric chloride solution Leaf Dark Blackish brown TanninRhizome Blackish brown TanninStem Blackish brown Tannin

Iodine solution Leaf Blue’s brown StarchRhizome Blue’s brown StarchStem Blue’s brown Starch

Ammonia solution Leaf Dark yellowish brown AnthraquinoneRhizome Yellowish brown AnthraquinoneStem Light Yellowish brown Anthraquinone

Aqueous Potassium hydroxide Solution (5%) Leaf Yellowish brown AnthraquinoneRhizome Yellowish brown AnthraquinoneStem Light Yellowish brown Anthraquinone

Table 3 Proximate analysis of Root, Stem and Leaf of Costus igneus

S.NO Parameters AmountRoot Stem Leaf

1 Total ash 4.2±0.0067c 2.2±0.026∗ ∗ 2.2±0.026 2 Acid insoluble 0.2±0.02 0.133±0.0731 0.167±0.022 3 Water soluble 0.74±0.0267 1.0±0.0266 1.0±0.0266 4 Moisture (%) (After 1 hour) 5.4±0.04b 10.4±0.1867∗ 14±0.427 5 Moisture (%) (After 1.5 hour) 7.4±0.393 14.366±0.0633 21.2±0.447 6 Crude fibre 7.767±0.0022 7.4±0.0267b 8±0.0067 7 Carbohydrate 9.31±0.0074a 7.87±0.0156b, ∗ ∗ 11.2±0.107 8 Protein 2.783±0.00166a 1.6±0.0156a, ∗ ∗ , 3.3±0.06

The results were expressed as mean ± Standard deviation; Statistical signifi-cance 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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small, thin walled compact cells (Fig.7B). The periderm is 150µm wideand the cortex is 400µm wide. The stele has about 10 exarch xylemalternating with equal numbers of phloem strands (Fig.7A). The stelehas distinct endodermis with cylindrical narrow cells; their inner andlateral walls have U-shaped thickenings. Inner to the endodermis is anarrow layer of hyaline cells, which are circular to rectangular in out-

line. The xylem strands are numerous and each strand has a widecircular, thick walled metaxylem elements and narrow proto xylemelements; the metaxylem is 60-70 µm wide (Fig.7C); the proto xylemelements are 40 µm wide. A prominent mass of phloem occurs inbetween the xylem. No sclerenchyma tissue is seen in the stele.

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phological and anatomical characters of leaf, rhizome and root amongthem Costus igneus have a characteristic anatomy that could be usedto distinguish it from other members of Costaceae. Costus igneusstudied, however, appear to be a homogenous entity united by seriesof leaf, rhizome and root anatomical characters, including epidermalcells, mesophyll cells, the sclerenchyma cells, vascular bundle, cor-tex, xylem, phloem, starch grains, stelar bundle. These futures oflamina, rhizome and root anatomy endorse. The finding of Oteng-Yeboah (1981) in the same species of Costus from West Africa coin-cides with our present study. Differences in vegetative anatomy

Proximate analysis

The result of fluorescent analysis, ash (total, acid soluble,water soluble) content, moister content, carbohydrate and protein ofCostus igneus leaf, root and stem are represented in Tables (1, 2 and3) respectively.

DISCUSSION

The Costaceae plants were identified to have different mor-

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Source of support: DBT, New Delhi (BT/PR10018/NNT/28/95/2007), Conflict of interest: None Declared

among such members of Costaceae as Costus and Tapeinochilus(Tomlinson, 1956) have received some emphasis in phylogenetic dis-cussion. The anatomical structures of leaf, rhizome and root of Costusigneus differentiated that of C. lucanusianus and C. afer that wasstudied by Edeoga and Okoli (1997). The leaf has two layers of thinepidermal cells and four layers of wide, tangentially oblong thin walledmesophyll cells. It is 300µm thick. Randomly distributed in the cortexare small circular cortical vascular bundles. The cortical bundles arecollateral with a wide, angular fairly thick walled xylem elements andsmall cluster of phloem elements. Thin roots have fairly wide superfi-cial sequent periderm and narrow homogenous paranchymatous cor-tex. The stele has about 10 exarch xylem alternating with equal num-ber of phloem strands. No sclerenchyma tissue is seen in the stele.Tomlinson (1956) presented some reasons for treating Costaceae as aseparate family from Zingiberaceae. The lamina, rhizome and rootanatomy of these Costus species investigated seems to agree withthe Tomlinson’s (1956) reasons for supporting the raising of the ge-nus to rank of family by Nakai (1941). Cytology, phytochemistry ofCostus igneus studies helps to clearly differentiate Costus igneusfrom other Costaceae plant. Tomlinson (1956) outlined some diagnos-tic features of the leaf, stem, rhizome and root anatomy in theZingiberales as a whole but did not specify how these could be usedin resolving critical systematic problem as in the presently investi-gated Costus igneus anatomical study. The proximate values showsthat the protein content is relatively low but it can contribute to theformation of hormones which controls a variety of body functionssuch as growth, repair and maintenance of body protein. The rela-tively high carbohydrate content can be used as energy source andalso it is necessary in the digestion and assimilation of other foods.The moisture and ash content is useful in assessing the quality ofgrading the plant and also gives an idea of the amount of mineralspresent in the samples. At present investigation, Costus igneus havea characteristic anatomy and proximate analysis that could be used todistinguish it from other members of Costaceae.

ACKNOWLEDGMENTS

Dr.T.Eevera acknowledges DBT, New Delhi (BT/PR10018/NNT/28/95/2007) for providing financial support for this project.

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