The drug evaluation and bioassay of traditional herbs of various herbal systems is now getting more momentum throughout the world. It is the high time now to evaluate scientifically the information stored in different herbal medicine systems of the world in terms of their pharmacognostic, phytochemical and pharmacological characterization. So knowledge of pharmacognosy of individual medicinal plant is very important aspect for herbal based drug discovery. Therefore in this study, an attempt has been made to evaluate such ethnomedicinally important plant, pharmacognostically along with antimicrobial and phytochemical analyses which are still unexplored. In this study different pharmacognostical parameters of Ipomoea cairica (L.) Sweet of Convolvulaceae, an ethno botanically important medicinal plant have been investigated. The plant is commonly known as ‘Railway creeper’ or Morning glory. The plants are medicinally used as an antioxidant, anti-inflammatory, antiviral and highly potent against malaria. Palisade ratio is 6. Leaves are amphistomatic. Stomatal index is 11. Trichomes show an assortment of both nonglandular, multicellular and glandular, multicellular, sessile types. These two types are recorded on both surfaces. Vessel elements are moderately long with simple and transverse or obliquely placed perforation plates. Pits are simple and tails are frequently present with some vessel elements. In the leaf extract, the detected phytochemical groups are alkaloids, flavonoids, steroids and triterpenoids, reducing sugars, tannins, gums and saponins, etc. In the stem extract flavonoids, steroids and triterpenoids, reducing sugars, tannins and saponins are present. Different histochemical localizations have been identified in the stem which contain some specific phytochemical groups like lignins, celluloses, suberins, proteins and alkaloids. Ash value and moisture content of the leaf are 35.88 % and 89.25 % respectively. The highest inhibition zone was found against Salmonella typhi ATCC 19430 and Klebsiella aerogenes W70 (inhibition zone, 15 mm) in the methanolic extract. Ethyl acetate extract showed the maximum inhibition on Pseudomonas aeruginosa NCTC10662 (inhibition zone, 19 mm). This study will be very helpful to herbalists and pharmacologists for proper evaluation and validation of folk drug.
Keywords: Pharmacognostic analysis; phytochemical screening; antimicrobial activity; micromorphology and anatomy; Ipomoea cairica; ethnomedicinally important plant
The role of pharmacognostic and anatomical data for traditional knowledge of folklore medicine is highly important since the earlier past. Use of micromorphology and anatomy is now a recognized tool in the field of pharmacognosy as well as plant systematics. Therefore in this investigation, an attempt has been made to evaluate this ethnomedicinal plant pharmacognostically, including its morpho-anatomy and antimicrobial activity. The leaves and stem of the investigated taxa have been considered here in this investigation because leaves and stems are commonly used by the tribal and common people for curing the diseases. Use of micromorphology and anatomy is now a recognised tool in the field of plant systematics [1, 2]. Importance of epidermal characters in general and those of trichomes in particular and comparative wood anatomy are widely recognised in taxonomic consideration of angiosperms [3, 4, 5, 6, 7, 8]. Ontogeny and structure of stomata are now also considered as an important taxonomic character for many of the angiospermic taxa [8, 9, 10, 11, 12, 13]. The members of different genera and families of angiosperm have been studied anatomically by various workers with special emphasis on leaf epidermal micromorphology [14, 15].Only to some extent, the ontogeny, structure of stomata and phytochemical studies of different members of Convolvulaceae have been studied by different workers. Chemical analysis and biological assays are very important aspects in pharmacognostic evaluation of medicinal plants [16, 17]. Along with various pharmacognostical evaluation of medicinal plants now there is an urgent need to study the antibacterial properties of medicinal herbs, which will be helpful in the treatment of several diseases caused by bacteria. With this scientific documentation, we now know why certain herbs are effective against specific conditions.
Common name: ‘Railway creeper’, Morning glory.
Botanical Characteristics: Perennial twiner with tuberous root-stock. Leaves palmately 5 to 7-partite; segments elliptic - obovate or lanceolate, narrowed at both ends, retuse, mucronate at apex, glabrous. Flowers in 1 to 3-flowered cymes. Calyxsegments unequal, ovate, mucronulate, tuberculate on the back of outer ones. Corolla 6 -7 cm long, white or purple. Capsules 2 -celled, 4 -valved. Seeds pubescent
Throughout the year.
Distribution: A native of Tropical Asia and Africa, now widely grown in tropical countries including India.
379 Habit and habitat: Perennial twiner with tuberous root- stock. Terrestrial, common among bushes, hedges of gardens, waste places and outskirts of forests. Also cultivated occasionally in the gardens, parks, railway platforms.
Parts used: Whole plant.
Medicinal uses: Antioxidant, antiviral, highly potent against malaria.
Chemical constituents: lignans, arctigenin, matairesinol and trachelogenin, indole alkaloids.
Intensive field work was conducted covering all the seasons so as to collect detail information on plant species found useful in ethnomedicine as well as for the other local uses of the plants occurring in Birbhum district in West Bengal. During etnobotanical surveys, all tribal localities, adjoining forest areas and the plains of the districts were visited. Routine methods of botanical collections and techniques of herbarium preparations were followed as suggested by [18]. Observations were made of the plant species with respect to their location, habit, habitat and other field characters.
For the study of foliar epidermis, leaf samples were cleared following the Bokhari’s method [19]. The cleared leaf samples were then mounted on the slide with a drop of 10% glycerine and 1% aqueous safranin solution and observed under the compound light microscope.
For wood elements study, the stem pieces of the plant were macerated following the standard method [20]; washed several times, teased with needles, stained in safranin, mounted on the slides with 10% glycerine and observed under microscope. The drawings of the stem xylem element characters were made with the camera lucida and measurements were taken with standardized ocular micrometer in each cases.
Histochemical study was carried out by cutting the section of fresh materials following the standard methods [21, 22, 23]. It is generally used for on spot determination of localized phytochemicals present in the cells.
Screening of antimicrobial activity was carried out by agar diffusion method. Nutrient agar medium was prepared by suspending nutrient agar (Merk) 20g/L in distilled water. The pH value of the media was adjusted to 7.0, autoclaved, and O allowed to cool up to 45 C. 50 μl of extract was poured in 6mm wells punched in test culture seeded assay plates. 24 h old bacteria cultures in nutrient broth were used to seed the assay plates containing NAM. Assay plates seeded with bacterial cultures were incubated at 37° C, for 24 hours. After incubation antimicrobial activity was determined by measuring the zone of inhibition Eight common human pathogenic bacterial strains (Bacillus subtilis ATCC 11778, Enterococcus faecalis ATCC 19433 , Escherichia coli NCTC 10418, Klebsiella aerogenes W70, Micrococcus luteus NCTC2665, Pseudomonas aeruginosa NCTC10662, Salmonella typhi ATCC 19430 and Streptococcus faecalis MTCC 439) were selected for this purpose. The minimum inhibitory concentration (MIC Assay) method was applied.
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i) Leaf: Herbaceous, palmately 5 to 7- partite, segments elliptic-obovate or lanceolate, narrowed at both ends, retuse, mucronate at apex, glabrous.
ii) Stem: Perennial twiner with tuberous root- stock, branched, older ones somewhat woody, greenish young part, whitish-brown older part.
General description and measurement of the epidermal cells, stomata and trichomes of the investigated plant have been represented in Tables 1, 2 and 3.
Epidermal cells are irregular in shape and the outlines are strictly wavy on both the surfaces. Cell size is 28.60 μm x 71.42 and 37.51 μm x 55.71 μm on the upper and lower surfaces respectively. Cell frequency of the upper and lower surfaces 2 2 is 1211.45 /mm and 1497.80 /mm respectively. Palisade ratio is 6 (Table-1; Figure 1- A, B, C, D, E).
Leaves are amphistomatic i.e. stomata are present on both surfaces of the leaf. On both surfaces paracytic stomata are predominant with few anisocytic and anomocytic type. Size of the stomata on the upper and lower surfaces is 28.60 μm x 21.42 μm and 28.60 μm x 53.60 μm respectively. Stomatal frequency of the 2 upper surface is 33. 04 /mm and 198.23 2 /mm on the lower surface. Stomatal index is 11 (Table-2; Figure 1 A, B, C, D, E).
Trichomes show an assortment of both nonglandular, multicellular and glandular, multicellular, sessile types. These two types are recorded on both surfaces. Size and frequency of the nonglandular, multicellular trichomes of the upper surface are 142.84 μm x 42.85 μm and 2 16.60 /mm respectively. On the lower surface their size and frequency are 2 124.98 μm x 44.04 μm and 22.03 /mm . Diameter of the multicellular glands is 42.85 μm on the upper surface and 28.60 μm on the lower surface. Frequency is 2 2 11.01 /mm and 16.60 /mm on the upper and lower surfaces respectively. Trichome index is 0.1 (Table-3; Figure 1- F, G, H, I, J).
General description and measurement of the type and size, pittations, perforation plates of vessel elements, side wall thickening of tracheids, fibre size and nature, etc. of the investigated plant have been represented in Table 4.
Vessel elements are moderately long with simple and transverse or obliquely placed perforation plates. Pits are simple and tails are frequently present with some vessel elements. Size of the vessel element is 450.60 μm x 66.06 μm; frequency is 2 29.35 /mm (Figure 2- A, B, C, D).
Tracheids are long with spiral sidewall thickening. Diameter of the tracheids is 2 16.07 μm; frequency is 22.02 /mm (Figure 2- E).
Fibres are typically libriform type and are extremely long. Ends are mainly blunt, but pointed endings are also recorded. Pits are present but lesser in number. Size of the fibre is 660.01 μm x 10.71 μm; frequency 2 is 44.05 / mm (Figure 2- F, G, H, I).
The transverse section through the internodes of stem shows the following anatomical features (Figure 3- a, b).
Epidermis: Epidermis consists of single layered cells of compact arrangement and covered with cuticle.
Cortex: The cortex is massive and consists of three distinct zones. The first zone is 381 hypodermis of few cells thick, lying just below the epidermal layer. Two to three layers of parenchyma cells are present beneath the hypodermal layer is called the middle cortex. A continuous, compactly arranged barrel shaped cells forms the starch sheath layer which is the last zone of cortex. Crystals and latex are present in some cells of the middle cortex.
Vascular bundles: Vascular bundles form a continuous cylinder of xylem and phloem. They are collateral, conjoint and open type with outer phloem, middle xylem and inner phloem patches. Intraxylary phloem patches are lying on the margin of the pith and are discontinuous.
Pith: Pith is massive with large parenchymatous cells. Middle portion of the pith is with many hollow cavities. Some crystal and latex containing cells are present in the pith region.
Colour: Dark green; Odour: Characteristic; Taste: Soft slimy feeling; Texture: Herbaceous, glabrous (in fresh form).
Through the phytochemical tests of the methanolic extract of leaf and stem, the important phytochemical groups have been detected in both the cases which actually confirm the medicinal properties of this plant. In the leaf extract, the detected phytochemical groups are alkaloids, flavonoids, steroids and triterpenoids, reducing sugars, tannins, gums and saponins, etc. In the stem extract flavonoids, steroids and triterpenoids, reducing sugars, tannins and saponins are present (Table 5, Figure IV).
Histochemical study has been carried out to detect various phytochemicals localized in different tissue zones of the stem. Different histochemical localizations have been identified in the stem which contain some specific phytochemical groups (lignins, celluloses, suberins, proteins and alkaloids) (Table 6).
a) Total ash - 35.88% b) Water soluble ash- 18.78% c) Acid insoluble ash- 05.90% ii) Moisture Content - 89.25 % (in fresh form).
Here in this study it is observed that drug powder treated with different chemical reagents gives characteristic colourations when seen under UV light and it is compared with the colourations observed under ordinary light. In some cases there are marked differences in colour (Table 7).
Methanolic foliar extract of Ipomoea cairica exhibited inhibitory action against Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella aerogenes, Micrococcus luteus, Salmonella typhi and Streptococcus faecalis. Ethyl acetate soluble foliar extract was able to inhibit all the selected bacteria.
The highest inhibition zone was found against Salmonella typhi and Klebsiella aerogenes (inhibition zone, 15 mm) in the methanolic extract. Ethyl acetate extract showed the maximum inhibition on 382
Pseudomonas aeruginosa (inhibition zone, 19 mm).
Salmonella typhi and Klebsiella aerogenes (inhibition zone, 15 mm) > Escherichia coli, Streptococcus faecalis and Micrococcus luteus (inhibition zone, 14 mm) > Bacillus subtilis (inhibition zone, 13.5 mm) > Enterococcus faecalis (inhibition zone, 12 mm) (Figure VI).
Only Salmonella typhi (inhibition zone, 10 mm) was inhibited by the water soluble foliar extract (Figure VI).
Pseudomonas aeruginosa (inhibition zone, 19 mm) > Salmonella typhi (inhibition zone, 16 mm). > Escherichia coli (inhibition zone, 15 mm) > Bacillus subtilis (inhibition zone, 14 mm) > Klebsiella aerogenes (inhibition zone, 12mm) > Enterococcus faecalis, Micrococcus luteus and Streptococcus faecalis (inhibition zone, 10 mm) (Figure VI).
Data presented in the tables are averages of 20 observations * 384
Dragendroff’s reagent Alkaloids Wagner’s reagent Alkaloids Shinoda’s tests Flavonoids 10% NaOH Flavonoids Salkowski test Steroids and triterpenoids Benedict’s reagent Reducing sugars Fehling’s reagent Reducing sugars Molish’s test Gums 10% aqueous Tannins potassium dichromate solution 10% aqueous lead Tannins acetate solution 5% aqueous ferric Tannins chloride solution 1% lead acetate Saponins Borntrager’s test Anthraquinone s - = Absent; + = Present Orange brown ppt ++ _ Orange brown ppt + _ Magenta colour + + Magenta colour ++ ++ Reddish-blue and + + green florescence Brick red ppt + _ Brick red ppt + + (Faint) Red-violet ring + _ Yellowish-brown + +++ ppt Yellow ppt +++ +++ Greenish-black +++ +++ colour White ppt ++ + _ _ _ Brownish to rose Yellowish brown Yellow to brown Yellow to brown Deep brown
cellulose Sudan III Suberin red Heating with Suberin Yellow strong H2SO4 Lugol’s solution Protein Millon’s reagent Protein Dragendroff’s Alkaloids reagent Wagner’s Alkaloids reagent Kedde reagent Glycoside - = Absent; + = Present sclerenchyma - + Hypodermis and xylem + Hypodermis and xylem ++ Parenchyma cells of cortex and pith ++ Phloem region, parenchyma cells of cortex and pith +++ Xylem and hypodermis +++ Xylem, some cells of cortex and outer phloem region _ _ _ different Indian systems of medicine and they are constantly being screened for their biological activities [25].
This is a fascinating area of research which has proved quite rewarding, the scientific examination of plants used for medicinal, narcotic and other purpose by the natives. Therefore in this investigation, an attempt had been made to evaluate such ethnomedicinally important plant pharmacognostically including morpho-anatomy and their
pharmacognosy, anatomy and antimicrobial activity which are lying still unexplored. The leaves and stems of the investigated taxa (Ipomoea cairica) had been considered here in this investigation because leaves along with the stems are commonly used by the tribal people and common folk for curing the diseases.
Pharmacognostic evaluation of leaf drugs are not so very expensive though this drug is of tremendous biomedical value.
16 A
14 12 10 8 6 4 2 0
B C
Bacillus subtilis Enterococcus faecalis Escherichia coli Micrococcus luteus Pseudomonas aeruginosa Salmonella typhi Streptococcus faecalis Bacillus subtilis Enterococcus faecalis Escherichia coli Micrococcus luteus Pseudomonas aeruginosa Salmonella typhi Streptococcus faecalis 388
389
00x
390
Lignans Matairesinol from plants which include macromorphology, anatomy, phytochemical and pharmacological studies. The major problem of the commercial supply of crude drugs is identification of genuine drug. Crude drugs may easily be adulterated or substituted by or confused for other ones because neither it has any trade name printed on it nor it carries any identifying structure for the easy identification of it by the plant taxonomists, rather drug samples supplied are shrunked, rolled, twisted, deformed and discoloured. So, pharmacognostic evaluation of crude drugs with macromorphology, micromorphology, organoleptic tests, ash value, histochemical colour reactions and UV fluorescence study will help in identifying genuine drugs and thus in checking adulteration, because the tests are very specific for a particular drug.
391
Here in this study all crude drugs in fresh as well as dried form obtained from respective investigated ethnomedicinal plant had been easily identified by some pharmacognostic characters like epidermal cells size, trichome types and index, stomatal type and index, palisade ratio, crystals, sclereids, size of fibres and vessel elements, ash value, UV fluorescence feature, etc. Palisade ratio is 6. Leaves are amphistomatic. Stomatal index is 11. Trichomes show an assortment of both nonglandular, multicellular and glandular, multicellular, sessile types. Ipomoea cairica having the wavy epidermal cell on both the surfaces (upper surface, 28.60μm x 74.42μm; lower surface, 37.51μm x 55.71μm). Though stem anatomy of the investigated plants is found to be very common to each plant, although it is specific in case of number of layers of some tissues present in cortex, vascular zone and pith and by using these features investigated plants can be distinguished along with other anatomical features like vessel elements, fibres, tracheids of xylem. Vessel elements are very much variable in size found in angiosperms and it is used as identifying character in identification of plants [26]. In this study, vessel elements are found in various sizes; perforation plates are simple, oblique or transversely placed. Fibres are typical libriform type with pointed or blunt tips. Presence or absence of fibre-tracheids has considered as a distinct feature here in this study for identification of investigated plants. Diameter of tracheid is found variable in many cases. Considering all those xylem elements features it has been found that they are very important one in identification of each plant of this study and also be helpful in authentication of crude drugs obtained from those plants. Through the phytochemical tests of the methanolic extract of leaf and stem, the important phytochemical groups have been detected in both the cases which actually confirm the medicinal properties of this plant. In the leaf extract, the detected phytochemical groups are alkaloids, flavonoids, steroids and triterpenoids, reducing sugars, tannins, gums and saponins, etc. In the stem extract flavonoids, steroids and triterpenoids, reducing sugars, tannins and saponins are present Different histochemical localizations have been identified in the stem which contain some specific phytochemical groups (lignins, celluloses, suberins, proteins and alkaloids).
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In case of crude drug identification ash value plays a very important role which includes % of total ash, water soluble ash and acid insoluble ash [27]. Here in this investigation ash value for each plant is very distinct and different from one another. It gives a marker character for identification of crude drugs obtained from those investigated taxa. Ash value and moisture content of the leaf are 35.88 % and 89.25 %respectively. Now the traditional medicinal plants are being screened on various lines of bioassay for their antimicrobial activity [28, 29, 30]. Here in this investigation, the leaf extracts of different solvents (methanol, water and ethyl acetate) of the plant have been tested for antimicrobial activity against eight Gm (+ve) and Gm (-ve) bacteria i.e., Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella aerogenes, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella typhi and Streptococcus faecalis. The highest inhibition zone was found against Salmonella typhi ATCC 19430 and Klebsiella aerogenes W70 (inhibition zone, 15 mm) in the methanolic extract. Ethyl acetate extract showed the maximum inhibition on Pseudomonas aeruginosa NCTC10662 (inhibition zone, 19 mm). Similarly fluorescence characters of the crude drugs are considered very important marker in making distinction among the drugs. Here some distinct features have been identified which are very much distinctive in identifying the respective drugs.
Thus, it may conclude that the present study will add some specific criteria for authentification of all those respective crude drugs and will be helpful for the quality control and to increase the economic potentiality of these drugs.
We are very grateful to the Head of the Botany Department, Visva-Bharati for providing the necessary laboratory facilities and also very thankful to the University Grant Commission, Govt. of India for financial assistance.