Pharmacognostical and Preliminary Phytochemical Studies on the Aerial Parts of Rubus racemosus (Roxb)

P.R. Kumar¹* and V. Vaidhyalingam²

¹Department of Pharmaceutical Sciences, Vels College of Pharmacy, Vels University, Pallavaram – 143.

²K. K. College of Pharmacy, 1/161, Sankaralinganar Road, Girukambakkam, Chennai-602 101.

ABSTRACT:

Rubus racemosus is commonly known as cheethi in Tamil. Traditionally it is used as an antidiabetic, abortifacient, muscle relaxant and free radical scavenging agents. Infusion of leaves was administered to stop diarrhea and some bleeding. There was no report on the pharmacognostic study of plant and hence the present investigation deals with anatomical, microscopical, fluorescence analysis, quantitative microscopy, determination of leaf constants, physiochemical, thin layer chromatography and high performance thin layer chromatography. Phytochemical studies showed the presence of flavanoids, saponin, terpenes, glycosides and phenols.

KEYWORDS: Rubus racemosus, Transverse Section, TLC, HPTLC

INTRODUCTION:

The straggling shrub Rubus racemosus belongs to the family Rosaceae. It occurs as decidious shrub; Subshrub; tender parts glandular; prickles recurved. Leaves odd-pinnate, to 12(16) x 8 (10) cm, chartaceous; margin- serrate; petiole to 5 (7) cm; stipules adnate to petiole, to 6 mm, persistent; terminal leaflet ovate, acute, to 8 x 6 cm, often sublobulate; laterals ovate-lanceolate, 7 x 3.5 cm. Inflorescence axillary, a few-flowered; peduncle 2 cm. Flowers 1 cm wide; pedicel to 1 cm; bracts subulate, 6mm. calyx-tube shallowly cup-shaped, with glandular hairs; lobes 5, ovate-acuminate. Petals 5, red, longer than sepals. Stamens α.Ovary glabrous; ovule. Fruits globose, 1 cm wide purple^1-2. Decoction of the root is useful for relaxed bowel and dysentery³. Family Rosaceae is known as a source of folk medicine for treatment of nervous disorders⁴.

MATERIAL AND METHODS:

The aerial parts of Rubus racemosus were collected from Nilgiri Hills in the month of August in the year 2006. The plant was authenticated by Dr.S.Rajan, Field Botanist, Survey of Medicinal Plants and Collection Unit, (Central Council for Research in Homoeopathy), Department of AYUSH.

A voucher specimen is deposited in the department of pharmacognosy. C.L.Baid Metha College of Pharmacy. Fresh plants were used for macroscopic and microscopic studies. The plant parts were fixed in FAA and dehydrated with graded series of tertiary butyl alcohol⁵. The sections were stained with toluidine as per the method⁶. Fluoroscence analysis was done by standard procedure⁷. TLC was followed using standard procedure from wagner⁸. Quantitative microscopy⁹, Determination of leaf constants¹⁰ were followed according to standard procedure. Physiochemical standard were done according to standard procedure¹¹. HPTLC was followed using standard procedure.

RESULTS AND DISCUSSION:

Macroscopy of the leaf:

Colour of the leaf is green, size 3 – 5 cm in length 1.5 – 2.5 cm in width, Shape-Oval, Petiole- small petiole, Margin-Serrate, Apex-Acute, Base-Symmetrical, Veins-6 – 8 veins on each side

Microscopical characters:

Anatomy of the leaf:

The leaf has prominent midrib and lateral veins and their lamina (Fig.1).

[GT – Glandular trichome; La – Lamina; LV – Lateral vein; MR – Midrib; NGT – non glandular trichome; VB – Vascular bundle].

Fig.2 Anatomy of the Leaf

[AbE – Abaxial epidermis ; Abs – Abaxial side; Ads – Adaxial side; Ep – Epidermis; GT – Ground tissue; La - Lamina; LV – Lateral vein; MR – Midrib; Ph – Phloem; PM – Palisade mesophyll; SM – Spongy mesophyll; VB – Vascular bundle; X - Xylem]

The leaf has dense epidermal trichomes on the adaxial side and smooth on the abaxial side. Young leaves are plicate longitudinally and the mature leaves are flat (Fig.2).

Midrib: The midrib has concave adaxial side and broad and thick abaxial part. It has the length of 1.6mm vertically and 1.75 mm horizontally. The epidermal layer is thin, made up of small, thick walled cells. Two or three layers inner to the epidermis are collenchymatous, rest of the ground tissue has thin walled, circular, compact parenchyma cells. The vascular bundle is single, broad and bowl shaped. It consists of several long, radial rows of xylem elements and several small groups of phloem elements.

[B – Body of the trichome; Ep – Epidermis; GT – Ground tissue; LV – Lateral vein; MR – Midrib; NGT – Non-glandular trichome; Ph – Phloem; St – Stalk cells; X - Xylem].

Lamina: The lamina is 120 μm thick. The adaxial side is even and smooth; the abaxial side is undulate and hairy. The adaxial epidermis is thick with large squarish cells and thick cuticle; the cells are 20 μm thick. The abaxial epidermis is thin comprising of narrow, rectangular cells. The mesophyll tissue consists of two layers of short, thin palisade cells and 6-8 layers of small, lobed spongy parenchyma cells. The vascular bundles of the veinlets are small, surrounded by a layer of dilated hyaline bundle-sheath cells and narrow adaxial and abaxial extensions.

Trichomes: The leaf has dense glandular and nonglandular trichomes. The non glandular trichomes are unicellular, unbranched, thick walled and pointed at the tip. They arise from a pedestal of a group of cells raised above the level of the epidermis. The non glandular trichomes are 250-650 μm long.

[AdG – Adaxial groove; Col – Collenchyma; Ep – Epidermis; LB – Lateral bundle; AB – Abaxial bundle; MB – Median bundle; Pa – Parenchyma cells; Ph – Phloem; Sc – Sclerenchyma cells; Tr – Trichome; X – Xylem].

The glandular trichomes are more complex in structure. They occur on the leaf and the petiole or stem. The glandular trichomes have a long filamentous stalk and prominent, spherical head. The stalk consists of a single vertical row of cells or multiseriate elongated cells. The head of the gland has darkly staining compact mass of cells. The gland varies in length from 120 μm to 1.3 mm. The head is 50-120 μm thick.

Petiole (Fig.4): The petiole is circular in sectional outline and has shallow adaxial groove. It is 1.75mm in diameter. The upper part (distal part) of the petiole has a broad main, medianly placed vascular bundle and three accessory lateral bundles on either side, which diminish in size towards adaxial part. The lower part (proximal part) has wider median lervelle, two prominent lateral bundles and two smaller less prominent bundles (one set on either side of the adaxial part). All the bundles are collateral with thick band of sclerenchyma cells abutting the phloem tissue. The petiole has their epidermis with small thick walled cells. Inner to the epidermis is a narrow zone of two or three layers of collenchyma cells. The remaining ground tissue is homogeneous and parenchymatous (Fig.5).

Stem (Fig.6): The stem is uneven in outline with ridges and furrows due to the presence of thick epidermal trichomes. The stem has a thin layer of epidermis comprising of small thick walled cells. Epidermis is followed by about five layers of collenchyma cells. Inner to the collenchyma zone is a narrow cortical zone of parenchyma cells, some of them having chloroplasts.

[AB – Adaxial bundle; AdG – Adaxial groove; Col – Collenchyma; Ep – Epidermis; LB – Lateral bundle; MB – Median bundle; Pa – Parenchyma cells; Ph – Phloem; Sc – Sclerenchyma cells; X – Xylem].

[Co – Cortex; Col –Collenchyma cells; Ep – Epidermis; MR – Medullary – ray; Ph – Phloem; Pi – Pith; Sc – Sclerenchyma; St – Stele; X - Xylem].

The vascular cylinder is thin and continuous and consisting of several wedge-shaped vascular bundles placed close to each other. The vascular bundles are collateral and have thick mass of sclerenchyma caps, wide zone of phloem and radial files of xylem. The sclerenchyma cells are thin walled and wide lumened. Xylem elements are circular and thick walled. The pith is wide and parenchymatous.

[Co – Cortex; Col – Collenchyma; H – Head; Pi – Pith; St – Stalk].

Trichome Morphology (Fig.7 and 8):

The powdered sample of the leaf shows the trichomes and epidermal fragments. The non glandular trichomes are seen randomly distributed on the fragments of the epidermis. They are long, whiplike, thickwalled with smooth surface; they are tapering at the ends. The glandular trichomes are long stalked with spherical head. The cells of the head portion are darkly staining.

Flourescence analysis: The powder of Rubus racemosus was treated with various reagents and visualized under ultra violet radiations. The observations are furnished in Table No.1.

Table: 1

Material of treatment	Observation under UV light
Powder	Green
Powder + Water	Green
Powder + Conc.H2SO4	Dark green
Powder + Conc.HNO3	Pale green
Powder +Conc.HCl	Dark green
Powder + dil.H2SO4	Light green
Powder + dil.HNO3	Light green
Powder + dil.HCl	Green
Powder +1N NaOH	Blackish green
Powder + Ac.anhydride	Emerald green
Powder + Acetone	Dark green

Quantitative microscopy:

Quantitative microscopy was carried out and observations are recorded in Table no. 2

Table 2

The length of trichomes of Rubus racemosus is
Range	165µ Minimum	320.18µ Average	622.5µ Maximum
The width of trichomes of Rubus racemosus is
Range	12.7µ Minimum	34.82µ Average	41.4µ Maximum

Determination of Leaf Constants:

Determination of Leaf Constants were carried out and observations are recorded in Table no 3.

Table 3:

The range for the vein islet number of Rubus racemosus is
Range	2 Minimum	3 Average	4 Maximum
The vein let termination number of Rubus racemosus is
Range	5 Minimum	7 Average	9 Maximum
The range for the stomatal number of Rubus racemosus is adaxial surface
Range	4 Minimum	6 Average	8 Maximum
The stomatal index of Rubus racemosus in adaxial surface
Range	6.7 Minimum	11.2 Average	14 Maximum
The range of stomatal number of Rubus racemosus in abaxial surface
Range	2.5 Minimum	3.75 Average	5 Maximum
The range for stomatal index of Rubus racemosus in abaxial surface
Range	5 Minimum	3.7 Average	2.2 Maximum

Physiochemical standards:

The physiochemical standards like ash values,extractive values were carried out and observations are recorded in Table no 4.

Table 4:

S. No.	Parameters	Result
1.	Total ash	2.97% w/w
2.	Acid insoluble ash	1.80% w/w
3.	Sulphated ash	2.65% w/w
4.	Water soluble ash	0.18% w/w
5.	Alcohol soluble extractive value	8.35% w/w
6.	Water soluble extractive value	4.21% w/w

Preliminary Phytochemical Analysis:

The preliminary phytochemical analysis of the aerial parts of the Rubus racemosus is tabulated in Table no.5

Table 5:

Phytoconstituents	Petroleum Ether	Ethyl Acetate	Chloroform	Methanol	Aqueous
Alkaloids
Mayer’s	-	-	-	-	-
Hager’s	-	-	-	-	-
Wagner’s	-	-	-	-	-
Dragendroff’s	-	-	-	-	-
Carbohydrates
Molisch	+	+	+	+	+
Fehling’s	+	+	+	+	+
Benedict’s	+	+	+	+	+
Barford’s	+	+	+	+	+
Steroids
Liberman	-	-	-	-	-
Burchard Test	-	-	-	-	-
Sterols
5% potassium Hydroxide	-	-	-	-	-
Proteins
Biuret	-	-	-	-	-
Millon’s	-	-	-	-	-
Phenols
Ferric chloride	+	+	+	+	+
10% sodium Chloride	+	+	+	+	+
Tannins
10% lead acetate	+	+	+	+	+
!0 % sodium Chloride	+	+	+	+	+
Aqueous bromine Solution	+	+	+	+	+
Flavanoids
Amylalcohol + Sodium acetate + Ferric chloride	+	+	+	+	+
Gums and Mucilage	-	-	-	-	-
Glycosides
Glacial acetic acid + ferric chloride + conc. H2SO4	+	+	+	+	+
Saponins
Foam test	+	+	+	+	+
Terpenes
Tin + Thionyl Chloride	+	+	+	+	+

Thin layer chromatography:

TLC of the methanolic extract was developed in n butanol:aceticacid:water (4:1:5) showed one spot at an Rf value 0.45 and with chloroform:ethylacetate (6:4) showed one spot at an Rf value 0.59.HPTLC finger print of the extract showed 8 spots with an Rf value of 0.07, 0.08, 0.27, 0.39, 0.54, 0.59, 0.67 and 0.81. at 5µl.

CONCLUSION:

The present data generated would help in the authentication of the drug. The aerial parts of the plant Rubus racemosus were anatomically studied and reported first time. The spots observed in TLC and HPTLC fingerprint profile will be definitely useful for deciding the purity, quality of the drug to lay down standard.

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Received on 14.06.2010

Accepted on 07.07.2010

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 2(5): Sept.-Oct. 2010, 377-380