Received by Lynn Received on 2012-3-26
ID No. B175 Revised on
Pharmacognostic and pharmacological evaluation of Ruellia tuberosa L.
Rehmanullah, Muhammad Ibrar and Ishfaq Hameed
Department of Botany, University of Peshawar, Pakistan
Ruellia tuberosa Linn. was studied to investigate the macro – and
microscopical, vein islet and vein termination numbers, palisade ratio, stomatal index and different chemical parameters. The antibacterial, antifungal and phytotoxic activities of the crude extract of the plant were also determined. Five bacterial species were used. Among them Salmonella typhi, Escherichia coli, and Pseudomonas
aeruginosa were the most susceptible bacterial species having MICs 10, 4.0, and 14 mg/ml, respectively. Among the tested fungal species Fusarium solani, and
Aspergillus niger were more susceptible to crude extracts with MICs 1.34, 2.78, and 1.45 µg/ml, respectively. At the concentration of 10µg/ml the methnolic extract exhibited significant activity, at 100µg/ml the activity was good and at 1000 µg/ml the activity was moderate against Lemna minor. The above selected plants were
shown by in vitro assays to be a potential source for natural antifungal, antibacterial and phytotoxic agents.
Keywords: Ruellia tuberose, Macro-microscopical study, chemical tests, antibacterial, antifungal, phytotoxic activities.
Morphology and histology makes the first step to get knowledge about the diagnostic features, which are ascertained through the study of the tissue and their arrangement, cell wall and cell content (Youngken, 1950). Plant anatomy has been a
source of fascination and a field of scientific inquirysince the time of the earliest
microscopists. The subject matterof plant anatomy centers on aspects of structure that
can beobserved not only with the light microscope but also using transmission and
scanningelectron microscopy and other tools of cell biology (Dengler, 2002). Not all the chemical compounds elaborated by plants are of equal interest to the pharmacognosist. Until relatively the so-called "active" principles were frequently alkaloids or specific glycosides usually with pronounced pharmacological properties; these received special attention, and in large measure constituted the principle plant drugs of the allopathic system of medicine. Other groups such as carbohydrates, fats, and proteins are of dietetic importance, and many such as starches and gums are used
in pharmacy but lacked any marked pharmacological actions (Evans, 2002). Putiyanan et al. (2009) reported that the macroscopic characters were studied for sample collecting and microscopic characters of transverse section of Fak khaao’s
leaves were compared to the leaf powders showing the upper and lower epidermis, trichome, collenchyma, palisade mesophyll, spongy mesophyll, stoma (guard cell), vascular bundles, etc., which were similar to microscopic description of drug powders. The values of stomatal index, veinlet termination number, vein-islet number and palisade ratio were calculated for standardization of samples which were 11.84?1.77, 5.95?1.31, 2.38?0.40 and 4.49?0.73, respectively. Latha and Kannabiran (2006) reported the presence of tannins, saponins, flavanoides, phenolic compounds, cardiac glycosides and carbohydrates in S. trilobatum. Ruellia tuberosa is used in folk
medicine due to its diuretic, diabetic, antipyretic, analgesic, and antihypertensive properties (Chiu & Chang, 1995). Recently, it is also being used as one of the components in a herbal drink in Taiwan (Chen et al. 2006). As no such literature was
available on this plant therefore the present study was carried out. Materials and Methods
The fresh specimens of the plants were collected from the Department of Botany, University of Peshawar, Pakistan. The plants were identified with the help of Flora of Pakistan by Prof. Dr. Abdur Rashid Plant Taxonomist (Ali and Qaiser, 2007). The specimen was cleaned and washed and dried in air for 15 days and was used for different tests i.e microchemical tests. These entire specimens were ground with the help of electric grinder and were mesh to 60 and were preserved in airtight bottles. Some fresh specimens were used to study morphological characters, while some of it was utilized for different anatomical parameters like vein islets numbers, vein termination number, palisade ratio, general anatomy of the root, stem and leaf and stomatal study. The macroscopical features of the plant were determined by following Wallis, (2004). The anatomy of the root and stem was determined by following a standard method of (Puruis et al. 1966). For leaf anatomy Subrahmanyam, (1996)
method was used. The vein islet numbers, vein termination number, palisade ratio was determined by following Evans, (2002). Different chemical parameters like alkaloids, mucilage, anthraquinon derivatives, calcium oxalate, tannin, lignin, starch, fats & oil, cutin, cellulose were determined by following Evans, (2002) and protein was determined by following Johnson, (1940).
Preparation of Extracts
Whole plants of Ruellia tuberosa L. was ground to 60 mesh powder using an
electric grinder. Fifty grams of each sample was separately soaked in 250 ml 70 % methanol for 72 hours. Thereafter each plant extract was passed through Whatman filter paper No. 1823. This process was repeated for 3 times. Evaporating in a rotatory
oevaporator at 40 C was carried out to concentrate the extracts. These extract was
ostored at 4 C priors to use. The methanolic extract and the standard drug were dissolved in dimethylsulphoxide (DMSO) at the concentration of 2 mg/ml and 1 mg/ml for antibacterial, 24 mg/ml and 1 mg/ml for antifungal, 30 mg/ml and 1 mg/ml for phytotoxic activities, respectively.
The tested bacteria, fungi and insects
Both gram negative and gram positive bacterial species Escherichia coli,
Salmonella typhi, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsella
pneumoni have been used. Nutrient agar medium was used for the growth of bacteria for agar diffusion method (Mariam et al., 1993) and nutrient broth medium for serial
dilution method (Spooner and Sykes, 1972). Fungal species Fusarium solani,
Aspergillus flavus, Aspergillus niger, Candida albicans and Candida glabarata.
Sabouraud dextrose agar (SDA) was used for the growth of fungi for agar dilution method following Mariam et al. (1993).
Antibacterial, antifungal and phytotoxic activities of the plant were determined by following a method of Ahmad et al. (2009).
Table No.1. Macroscopical features of the different parts of Ruellia tuberosa L.
sPlant Parameters Fresh Dry
Root Colour Brownish Brownish
Odour Characteristic Indistinct
Shape Cylindrical Cylindrical
Rootlets Present Present
Direction of growth Vertical Vertical
Fracture Fibrous Uneven
Texture Smooth Smooth
Stem Colour Dark Green Light Green
Odour Indistinct Indistinct
Shape Angular Angular
Phyllotaxis Opposite Opposite
Kind Herbaceous Herbaceous
Direction of growth Upright Upright
Fracture Uneven Fibrous
Texture Hairy Hairy
Leaf Colour Upper surface dark green Both surfaces light
lower surface light green green
Dimension Length is cm 2.3 cm, width is Length is cm 2.2 cm,
cm 1.1cm width is cm 1.0 cm
Incision Nil Nil
Composition Simple Simple
Venation Reticulate unicostate Reticulate unicostate
Margin Wavy Wavy
Apex Obtuse Obtuse
Surface Pubescent Pubescent
Shape of leaf Cordate Cordate
Table 2. Anatomical features of the different parts of the Ruellia tuberosa L.
Plant cell Value Root Stem
L(µm) W(µm) L(µm) W(µm)
22 10 34 27 Epidermis Minimum
38 19 55 39 Maximum
26 12 39 31 Mean
12 9 18 13 Xylem Minimum
32 15 38 17 Maximum
22 11 27 14 Mean
34 20 39 29 Phloem Minimum
56 28 55 41 Maximum
46 22 45 37 Mean
18 9 22 18 Endodermis Minimum
34 15 34 23 Maximum
26 11 28 20 Mean
14 10 16 12 Pericycle Minimum
26 16 27 20 Maximum
16 12 21 15 Mean
10 7 - - Pith Minimum
15 11 - - Maximum
13 9 - - Mean
14 8 18 12 Cortex Minimum
19 15 26 19 Maximum
16 12 23 15 Mean
Table 3. Microscopic Characteristics of the Ruellia tuberosa L. S. No Parameters Values
25.63 – 28.28 1 Vein Islet Number
55.21 – 58.43 2 Vein Termination Number
42.65 – 49.75 3 Palisade Ratio
14.23 – 15.42 4 Stomatal Index (Upper surface)
24.23 – 26.45 5 Stomatal Index (Lower surface)
Table 4. Microchemical screening tests of the different parts of Ruellia tuberosa L.
Plant Alk Muc Anth Cao Sap Tan Sta Fat Pro Lig Cut Cel part
Root + - - + - + + - + - - +
Stem + + - + - + + - + - - +
Leaf + + - + - - + - + + - +
Flower + + - + - - + - + + - +
Table 5. Antibacterial activity of the methanolic extract of the whole plant of Ruellia tuberosa L.
S. No Name of Fungus Linear growth Linear growth % Inhibition
of sample of control
1 Salmonella typhi 6 33 81.8
2 Pseudomonas aeruginosa 12 63.6
3 Escherchia coli 9 72.7
4 Staphylococcus aereus 17 48.4
5 Klebsella pneumoni 26 21.2
Table 6. Antifungal activity of the methanolic extract of the whole plant of Ruellia tuberosa L.
S. No Name of Fungus Linear growth Linear growth % Inhibition
of sample of control
1 Aspergillus niger 7 45 84.4
2 Aspergillus flavus 35 22.2
3 Fusarium solani 15 66.6
4 Candida albicans 20 55.5
5 Candida glabarata 30 33.3
Table 7. Phytotoxic activity of the methanolic extract of the whole plant of Ruellia tuberosa L.
Plant name Test plant Conc. of compound % growth
Ruellia tuberosa Lemna minor 100 67.25
Results and Discussion
Colour of the root was brownish in both fresh and dry forms. Odour was characteristic in fresh and indistinct in dry form. Shape was cylindrical in both fresh and dry forms. Rootlets were present in both fresh and dry forms. Direction of growth was vertical downward, fracture was fibrous in fresh and uneven in dry form, texture was smooth in both fresh and dry forms. Yadav et al. (2007) the macroscopical
characters like shape, colour, fracture, surface and taste of the root of the Chenopodium album Linn. Colour of the stem was dark green in colour in fresh and
light green in dry form. Odour was indistinct, shape was angular and phyllotaxis of the leaf on the stem was opposite in both fresh and dry form. Kind was herbaceous, direction of the growth was upright and texture was hairy in both fresh and dry forms. Fracture was uneven in fresh and fibrous in dry form. Badami et al. (2007) reported
the macroscopical parameters like colour, taste, odour, surface texture and shape of the heartwood of Caesalpinia sappan. Colour of the upper surface of the leaf was
dark green and that of the lower surface light green in fresh form and in dry form both surfaces were light green in colour. The length and width of the leaf was 2.3 and 1.1 cm in fresh and in dry form the length and width was 2.2 and 1.0 cm respectively. Composition of the leaf was simple in both fresh and dry form. Incision was nil in both fresh and dry forms. In both forms reticulate and unicostate venation was present. Apex was obtuse, surface was pubescent, and shape of the leaf was cordate in both fresh and dry form. Vidhu et al. (2007) reported that leaf of the Solanum nigrum is
simple, 2.5 – 8.5 cm long and 2.5 cm wide, ovate, or oblong, sinuate, toothed or lobed, narrow at both ends, petiolate, thin and pubescent. Dorsal surface is green in colour but the ventral surface is slightly pale. Venation is reticulate. The odour is characteristics and the taste is bitter (Table 1).
Epidermis of the root is rectangular in shape and is compactly packed. The mean length of the cells 26µ; mean width is 12µ. It is then followed by the cortical tissue. Mean length and width of the cell is 16µ and 12µ. Inner to the cortex is endodermis. Mean length and width are 26µ and 11µ. Single layer of pericycle is present, mean length and width of which is 16µ and 12µ. Xylem mean length and width are 22µ and 11µ. Mean length and width of phloem is 46µ and 22µ. In the center of the vascular bundle pith is present, mean length and width of which is 13µ and 9µ. Epidermis of the stem is spherical in shape and is compactly packed. The mean length of the cells 39µ; mean width is 31µ. It is then followed by the cortical tissue. Mean length and width of the cell is 23µ and 15µ. Inner to the cortex is endodermis. Mean length and width are 28µ and 20µ. Single layer of pericycle is present, mean length and width of which is 21µ and 15µ. Xylem mean length and width are 27µ and 14µ. Mean length and width of phloem is 45µ and 37µ. Epidermal cells of the leaf on adaxial surface are rectangular to hexagonal and polyhedral with smooth walls. The abaxial wall is irregular with undulating walls. Size of the epidermal cells; adaxial 133.75µ x 40µ. The abaxial one forming a network with the subsidiaries cells. Stomata is anisocytic type on abaxial side and staurocytic type on
adaxial side. Size of stomatal comples; adaxial one, 148.5 µ x 59.5 µ, abaxial one 90 µ x 68.5 µ, aperture size, adaxial one is 20.5µ (Table 2). Ferris et al. (2002) reported
co-efficient of variance, stomatal density, stomatal index, epidermal cells area and number of epidermal cells per leaf. Kanwal et al. (2006) reported parenchyma cells,
fibers, vessels, needle like elongated crystals and oil droplets in Pongamia pinnata.
Khan et al. (2001) reported epidermal cells, collenchyma, tracheids and fibers in Cyrtomium caryotideum.
Vein islet number ranges from 25.63 – 33.28, vein termination ranges from
55.21 – 58.43, palisade ratio ranges from 42.65 – 49.75, stomatal number of the upper
surface of the leaf ranges from 14.23 – 15.42 and that of the lower surface ranges
from 24.23 – 26.45 (Table 3). Kumar et al. (2008) reported the vein islet number (13),
vein termination number (18) and stomatal index (3.6) of the Portulaca oleracea.
Abere et al. (2009) reported the palisade ratio, stomatal number and stomatal index of the upper and lower surfaces, vein islet number and vein termination number of Dissotis rotundifolia
Alkaloid, Calcium oxalate, starch, protein and cellulose were present in all parts of the plant. Mucilage was absent from root and was present in other parts of the plant. Anthraquinon derivatives, saponins, fats and cutin were absent from all parts of the plant. Tannin was present in root and stem and was absent from leaf and flower. Lignin was absent from root and stem and was present in leaf and flower (Table 4). Hadjiakhoondi et al. (2005) reported essential oils from three wild varieties of Mentha
longifolia. Udayakumar et al. (2005) reported protein from Tridax procumbens.
Ansari et al. (2007) reported saponins from Balanites aegyptiaca. Anke et al. (2006)
reported new proanthocyanidins from Rumex acetosa. Different proanthocyanidins, a
polymer fraction and the new phenolglycoside 1-O-β-D – (2, 4-Dihydroxy-6-
methoxyphenyl)-6-O-(4-hydroxy-3,5-dimethoxybenzoyl)-glucopyranoside were obtained.
Methanolic extract of the plant exhibited low activity against Klebsella
pneumoni, moderate activity against Pseudomonas aeruginosa and Staphylococcus
aereusand significant activity against Salmonella typhi and Escherchia coli. The
extract exhibited low activity against Aspergillus flavus and Candida glabarata,
moderate activity against Fusarium solani and Candida albicans and significant
activity against Aspergillus niger. At the concentration of 10µg/ml the methnolic
extract exhibited significant activity, at 100µg/ml the activity was good and at 1000 µg/ml the activity was moderate against Lemna minor.
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