Pharmacognostic and HPTLC Finger printing of Gardenia gummifera L.F.

 

P. Sudhakar, P. Ramachandra Reddy

Plant Anatomy and Taxonomy Laboratory, Department of Botany, Osmania University, Hyderabad – 500 007.

 

ABSTRACT:

Gardenia gummifera L.f. is a shrub belonging to the family Rubiaceae. The leaves are used in the treatment of ringworms, malaria. The present paper provides a detailed account of the pharmacognostical studies. The study includes macro and micro morphological characters including powder characteristics, organoleptic characters, HPTLC finger printing and preliminary phyto-chemical aspects. This study would help as an appropriate source for authentification of the present studied drug.

 

 

INTRODUCTION:

Gardenia gummifera L.f. commonly known as ‘Dikamli’ in Hindi, ‘Cumbi – gum tree’ in English and ‘Bikki’ in Telugu belonging to the family Rubiaceae. It is a shrub found found throughout the Deccan peninsula extending northwards to Bhundelkhand and parts of Bihar. Leaves subsessile , elliptic to oblong or obovate – oblong, obtuse or subacute, glabrous; flowers subsessile, calyx 1 cm., long, densely pubescent; corolla 3.2 -3.8 cm., long; fruit 2.5 – 3.8 cm., long, oblong or ellipsoid, with numerous longitudinal elevated lines and with a tout beak¹. The leaves are used in the treatment of ringworms, malaria².

 

In the present investigation the leaves are found ethnomedicinally useful for cattle’s eye infections. The leaf paste is mixed with goat milk and dropped into eyes of cattle weekly twice for prevention of inflammation of viral infections by Gonds of Warangal district.

 

It is observed that there is lack of pharmacognostic studies found on the present studied drug. Hence, an attempt has been made to work on its pharmacognostical studies to help its identification. Hence, the information can be immense use for pharmacognosists, drug manufactures and medicinal practitioners in the identification of authentic drug.

 

MATERIALS AND METHODS:

Gardenia gummifera L.f. plant material was collected from various locations of Warangal district, Telangana, India. Collected material was taxonomically identified and deposited in Herbarium Hyderabadense, Department of Botany, Osmania University, Hyderabad. The leaves are boiled, fixed in F.A.A. (Formaldehyde – Acetic acid – Alcohol), dehydrated through xylene – alcohol series embedded in paraffin wax.

 

The sections were cut at 10 – 12 μm on Optica 1090A rotary microtome, stained with crystal violet and basic fuchsin combination and mounted in canada balsam³. Epidermal peels were obtained by gently scraping and peeling by razor blade, were stained with saffranine and mounted in glycerine. The powder microscopy characters were studied by boiling the drug in distilled water, stained in saffranine and mounted with glycerine.  The photomicrography was done on Olympus BX-53 trinocular microscope attached with digital Sony camera.

 

High Performance Thin Layer Chromatography (HPTLC):

Successive extraction was carried out using Soxhlet apparatus. 25 g leaf powder of Gardenia gummifera L.f. was taken in Whatmans No.1 filter papers, packed individually and placed in Soxhlet thimble and 250 ml solvent was taken in each round bottom flask. The leaf powder was extracted successively with petroleum ether, chloroform, ethyl acetate, and methanol. The process of extraction continued for 7 hours or till the solvent in siphon tube of an extractor become colorless. The extracts were cooled, filtered through Whatmans No.1 filter paper and extractions were carried out in the order of increasing polarity of those solvents i.e. from petroleum ether to methanol and preceded for phyto-chemical screening⁴. After the extraction the solvents were removed using rotary evaporator (Heidolf LABAROTA Efficient Avaporators 4000) and crude residues were obtained. 

 

RESULTS:

Macroscopy:

Leaves subsessile, elliptic – oblong or obovate – oblong, obtuse or subacute, glabrous, base obtuse, acute or cordate, main nerves 12-18 pairs.

 

Fig. 1 Fruiting twig of G. gummifera

 

Fig.2 Leaf macroscopy of G.gummifera

Microscopy:

Leaf surface:

Epidermal cells 5-7 sided, few 4 sided, mostly polygonal anisodiametric, few isodiametric, rarely polygonal linear, sides thick, mostly straight to curved, few curved to wavy, surface smooth, contents dense with calcium oxalate crystals in few. Stomata anomocytic and paracytic, subsidiaries 2-5, monocyclic, guard cells reniform. Unicellular conical hair distributed all over, more on primary and secondary veins, irregularly arranged, variously oriented (Plate –) Epidermal cell frequency 2730 per sq. mm., stomatal frequency 50 per sq.mm., stomatal index 1.79 (Adaxial surface) (Plate -1A andB) and E.C.F. 1770 per sq.mm., S.F. 660 per sq.mm., S.I. 27.16 (Abaxial surface) (Plate -).  

 

Plate 1

 

PLATE -1:

1A. Leaf adaxial surface with stomata X 76; 1B. Leaf adaxial surface with unicellular conical hair X 92; 1C. Leaf adaxial surface X 132; 1D. Leaf abaxial surface unicellular conical hairs X 125; 1E. Leaf abaxial surface X 200

 

ABBREVIATIONS:

s-stomata; uch-unicellular conical hair; c- costal cells

 

Transverse section of leaf:

In T.S. leaf is ribbed on either sides but prominently on abaxial at midvein; lamina wings 145-170 (156) µm and midvein 990 – 1500 (1256) µm thick. Epidermis 1 – layered, cells over the lamina mostly barrel shaped, few tabular and oval to circular, adaxially larger, walls thin, contents scanty; cuticle thick, covered over the surface (Plate-2A,B).

Mesophyll dorsiventral with palisade and spongy tissues. Palisade mostly 2 – layered, at some places upto 3 layered, closely packed with small intercellular spaces, interrupted at midvein, secondary and tertiary veins; cells columnar, cylindrical, perpendicular to epidermis; 16 – 33 (25) µm long and 8 – 16 (11) µm wide, walls thin, interspersed with sphaerocrystals and prism in few, contents dense with chloroplasts. Spongy tissue 3-5 layered, cells circular, oval to oblong, spherical, dumbbell shape, elongated cells 19-36 (26) µm long 11-19 (15) µm wide, isodiametric cells 8-19 (14) µm in diameter; cells loosely arranged in a reticulum with large intercellular spaces, interspersed with crystals and dense with chloroplasts in few (Plate -2B).

 

Ground tissue of midvein contains collenchyma, parenchyma and sclerenchyma tissues. Sclerenchyma tissue is enclosing the central vascular cylinder and adaxial bundles. Collenchyma 6-9 layered in the adaxial ridge, 2-3 layered on the abaxial, angular or lamellar, contents scanty. Parenchyma 8-10 layered adaxially and 6 – 9 layered abaxially thick, cells polygonal, oval to circular and oblong, intercellular spaces narrow, with sphaeraphides in few. Sclerenchyma 2-3 layered, surrounding the central vascular cylinder (Plate-2A).

 

Vascular tissue consists of predominantly vascular cylinder at centre enclosing the 3-4 central medullary bundles besides 2 adaxial bundles. Central vascular cylinder 792 – 962 (857) µm and adaxial vascular bundles 68 – 88 (79) µm in diameter; conjoint, bicollateral, endarch. Phloem bundles are enclosed by endodermis. Few of sclerenchymatous cells are attached to one or two bundles. The adaxial bundles are endarch, collateral, enclosed by sclerenchyma towards abaxial side. Xylem consists of trachieds, vessels, fibers and xylem parenchyma arranged in radial rows. Tracheary elements numerous, thick walled, polygonal 14 – 27 (20) µm in diameter. Xylem parenchyma in between tracheary elements. In L.S. tracheary elements showing mostly helical and reticulate few scalariform and annular. Xylem surrounded by phloem on either sides consists of phloem parenchyma, sieve cells, fibers and companion cells. Centrally the ground parenchyma inside vascular cylinder consists of polygonal to spherical cells, thin walled with small intercellular spaces (Plate-2A).

 

Transverse section of petiole:

Subspherical, adaxially flat, 918-1339(1108) µm in diameter; covered by few unicellular conical hairs. Epidermis 1-layered, cells oval to spherical, barrel shaped, some cells containing crystals and chloroplasts in few, covered by thick cuticle. Collenchyma hypodermal, 4-8 layered, angular, contents slightly dense in some with crystals and chloroplasts in few. Parenchyma vertically 28-33 celled thick and laterally 24-27 celled thick, covering the entire hypodermal area beneath collenchyma, often interspersed with sphaeraphidalidioblasts (Plate-2C).

 

Vascular tissue consists of a single wedge shaped vascular bundle at the centre and 2-4 small adaxial bundles on either sides at adaxial side. The central vascular bundle is laterally 518 – 604(559) µm long and vertically 270 – 388(346) µm wide, endarch, conjoint, bicollateral. Xylem is made of vessels/tracheids, xylem parenchyma and fibers; tracheary elements numerous, arranged in radial rows and few laterally aligned, interspersed by xylem parenchyma and few fibers in between; xylem is surrounded by phloem on both sides, consists sieve cells, companion cells, phloem parenchyma and phloem fibers.

 

Central vascular bundle is enclosed by a 1-3 layered sclerenchyma, continuous towards abaxial side. The smaller adaxial bundles are conjoint, collateral, enclosed by a sclerenchymatous sheath. A small phloem bundle is present one on each side of the central wedge shaped bundle (Plate-2C ).

 

Plate 2

 

PLATE -2:

2A. T.S. of leaf midvein X 58; 2B. T.S. of leaf lamina X 185; 2C. T.S. of petiole X 40

 

ABBREVIATIONS:

e-  epidermis; ade – adaxial epidermis; abe – abaxial epidermis; pl – palisade; sp-spongy tissue; co-collenchyma; p-parenchyma;  scl – sclerenchyma; vc- vascular cylinder; avb- adaxial vascular bundle; vb-vascular bundle.

 

Powder microscopy:

Powder consists of pieces of epidermis with stomata; pieces of epidermal cells with straight to curved sides; numerous unicellular conical hairs; costal cells polygonal to linear with straight to curved sides (Fig.3 i,ii,iii ).

 

i. Trichome

 

ii. Piece of epidermis

 

iii. leaf epidermis with stomata

Fig. 3. i. Trichome; ii. Piece of epidermis; iii. leaf epidermis with stomata

 

Organoleptic characters:

Colour- Green; Touch-Coarse; Odour- Fragrant; Taste – No characteristic.

 

Preliminary phyto-chemical analysis:

The preliminary phyto-chemical screening in the present study revealed the presence of steroids and triterpenoids in ethyl acetate, chloroform and methanol extracts while carbohydrates and flavonoids were present in ethyl acetate extract. Phenols and tannins were present in methanol extract only. Cardiac glycosides and gums were present in petroleum ether and chloroform extracts. Saponins were present in methanol extract. Alkaloids are absent in all extracts respectively (Table-1).

 

 

Profile of HPTLC finger printing:

The profile of chromatographic separation of leaf methanol extract scanned at 254 nm, reveals five spots (Fig. 4) out of which spot 5 possess maximum composition with R_f at 0.94. While, densitogram scanned at 366 nm, revealed 8 spots with spots 3 and 2 showing maximum composition at R_f0.33 and 0.21 respectively(Fig.6).

 

It is evident from the data that these are characteristic for the studied drug, which will help in identification and authentication of the drug. This is considered as valuable standards in pharmacopoeia. At 254 nm, five spots appear at R_f 0.13, 0.33, 0.79, 0.82 and 0.94 (All brown) (Fig. 5) with various concentrations while at 366 nm, 8 spots appears at R_f, 0.09 (yellow), 0.21(blue), 0.33 (blue), 0.51 (blue), 0. 74 (yellow), 0.76 (yellow) 0.80 (yellow) and 0.95 (red) (Fig.7). This is a vital finger print parameter to ensure the reliability and reproducibility of drug.

 

 

Table-1:Preliminary phytochemical analysis of Gardenia gummifera

‘+’ = present; ‘-‘ =absent

 

Fig.4: HPTLC densitogram of methanolic extract of G.gummifera scanned at 254 nm by using chloroform: methanol (8:2 v/v)

 

Fig.5:High performance thin layer chromatography image G.gummifera at 254 nm in chloroform: methanol (8:2 v/v)

 

Fig.6:HPTLC densitogram of methanolic extract of G.gummiferascanned at 366 nm by using chloroform: methanol (8:2 v/v)

 

Fig.7: High performance thin layer chromatography image of G. gummifera at 366 nm in chloroform: methanol (8:2 v/v)

 

DISCUSSION:

The epidermal cells in surface are reported as 4-8 sided with undulated or sinuate sides on either surfaces^5,6. But presently epidermal cells are mostly 5-7 sided, few 4 sided, mostly polygonal anisodiametric to isodiametric with sides thick, straight to curved and curved to wavy on either sides, but not sinuate as observed earlier^5,6. Further, the epidermal cells are found to contain calcium oxalate crystals in few. The epidermal cells on the adaxial are more frequent on adaxial being 2730 per sq.mm, compared to the abaxial surface with 1770 per sq.mm. The costal cells are 6-7 sided, polygonal anisodiametric to linear with sides thick, straight to curved containing calcium oxalate crystals in few.

 

The stomata are reported as mostly paracytic and rarely anomocytic with subsidiaries mostly of f- type and rarely a-and c-type (Patil and Patil, 2011). Leaves are amphistomatic, with rubiaceous type of stomata⁵. Presently they are amphistomatic as observed earlier⁵ with anomocytic and paracytic stomata as also described earlier⁶ with subsidiaries indistinct of f-type on adaxial while mostly c-type and rarely a-type on abaxial. 

 

The trichomes have been described as unicellular with curve at the apex in Gardenia⁷ but presently they are unicellular conical hairs but without curve at the apex as reported. Further, they are dispersed all over with more on primary and secondary veins.

 

Review of earlier literature reveals that, there is no information on the leaf, petiole anatomy and Preliminary phyto-chemical and HPTLC finger printing of Gardenia gummifera. However, occurrence of rosette crystals along with small acicular crystals in Gardenia^6,7  which is also presently confirmed.

 

CONCLUSION:

The powder microscopic features and organoleptic characters along with the anatomical and HPTLC fingerprinting are diagnostic to establish the pharmacopoeial standards for the drug.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Dr. P. Padma Rao, A.R.O.(Pharmacognosy), Drug Standardisation Unit (CCRH), Hyderabad for valuable suggestions and encouragement, Shri. Y. Sairam, Vice President, Anchrom enterprises Pvt. Ltd., Mumbai, for HPTLC facility, Dr. P. Uday Kumar, Deputy Director, Pathology Division, National Institute of Nutrition, Hyderabad for the microtome facility and thankful to the Head, Department of Botany, Osmania University, Hyderabad for providing facilities.  

 

REFERENCES:

1.     Kirtikar KR, Basu BD. Indian medicinal plants. Bishen Singh Mahendra Pal Singh, Dehradun. 2012.

2.     Kumari P, Singh GS. Ethnomedicinal study of medicinal plants used by Taungya community in Terai Arc landscape. India. J. Ethnopharmacol. 2009; 123: 167-176.

3.     Johansen DA. Plant Micro technique. Mc. Graw Hill, Book Co., New York. 1940.

4.     Sethiya NK, Brahmbhat K, Chauhan B, Mishra SH, 2016. Pharmacognostic and Phyto-chemical investigation of Ensetesuperbum (Roxb.) cheesmanpseudostem. Indian J. Nat. Prod. Resour. 2016; 7(1): 51-58.

5.     Pant DD, Mehra B. Ontogeny of stomata in some Rubiaceae. Phytomorphology. 1965; 300-310.

6.     Patil, CR, Patil DA. Investigations on foliar epidermis in some Rubiaceae. Journal of Phytology. 2011; 3(12): 35-40.

7.     Metcalfe CR, Chalk L. Anatomy of the Dicotyledons, Vol. I and II, Clarendon press, Oxford. 1950, Vol. I andII.

 

 

 

 

Received on 16.08.2017       Modified on 19.09.2017

Accepted on 16.10.2017     ©AandV Publications All right reserved