Classical taxonomy studies of medicinally important Ipomoea leari.

BACKGROUND: Ipomoea leari which belongs to the family Convolvulaceae is an unexplored medicinal plant in the Indian medicinal system. According to ethnobotanical information, the whole plant is used for various disorders such as anti-inflammatory, psychotomimetic and anticancer activities. The current study seeks to standardize the parameters for this herb.
MATERIALS AND METHODS: The identification of the pharmacognostical, morphoanatomical characters of Ipomoea leari (leaf, stem and root) were carried out in terms of organoleptic, macroscopic, microscopic, physicochemical, florescence and phytochemical analyses. Physicochemical parameters such as total ash, moisture content and extractive values were determined by World Health Organization (WHO) guidelines. The plant material was fixed in formalin-5 mL + acetic acid-5 mL + 70% ethyl alcohol-9 mL (FAA) and dehydrated with graded series of tertiary-butyl alcohol. Toluidine blue, a polychromatic stain was used for staining the sections and then whole components were observed with Nikon lab photo device with microscopic units.
RESULTS: Microscopically, leaf consists of prominent midrib and the lamina, both having dorsiventral symmetry. The stomata are actinocytic. The stem consists of an epidermal layer of one cell thickness, wide cortex, vascular cylinder and wide pith. The root measuring 1.6 mm thick was studied. It consists of uniformly thick and continuous periderm, wide cortex and thick vascular cylinder. Qualitative analysis revealed the presence of carbohydrates, flavonoids, glycosides, steroids and phenols. The pharmacognostic studies were carried out in terms of macroscopic, phytoconstituent and chromatographic analyses of Ipomoea leari. Various standard methods were adopted to carry out the investigation.
CONCLUSION: The results of the present study provide valuable pharmacognostic information of Ipomoea leari for its identification. Our result's suggest that Ipomoea leari is a promising candidate as an adjuvant therapy in various disorders and preparation of monograph.

INTRODUCTION

Ipomoea is a genus of annual vines as well as evergreen shrubs and perennials. Ipomoea is large genus of twining, creeping, floating or erect herbs, shrubs or trees widely distributed throughout the tropical and warm temperate regions of the world. Several members of the genus Ipomoea are widely used in different parts of the world for the treatment of several diseases, such as, diabetes, hypertension, dysentery, constipation, fatigue, arthritis, rheumatism, hydrocephaly, meningitis, kidney ailments and inflammations. Some of these species showed antimicrobial, analgesic, spasmogenic, hypoglycemic, hypotensive, anticoagulant, anti-inflammatory, psychotomimetic and anticancer activities. Alkaloids, phenolic compounds and glycolipids are the most common biologically active constituents found in the plant extracts.[123456]

Ipomoea leari which belongs to genus Ipomoea and Family - Convolvulaceae, is a twining perennial vine. This plant is famous as Blue Dawn and morning glory vine with striking purple-blue color flowers [Figure 1]. The plant is commonly found in hilly regions of India such as Ooty, Nainital and Mussoorie, scrambling over fences and over woody plants. The flowers bloom in the morning and fade to magenta in the evening. Ethnopharmacological information available about the plant indicates that the root is used in conditions such as dysentery, blood pressure and Waker carcinoma.[7] To standardize quality control parameters we investigated the plant morphoanatomy closely before proceeding with further evaluation.

Figure 1

Whole plant of Ipomoea leari

There have been no reported pharmacognostic literatures available on the Ipomoea leari root, leaf or stem. Hence, in this direction the present investigation was conducted for evaluating the various parameters such as macroscopic, microscopic characters and phytochemical parameters of the Ipomoea leari for the determination of raw material impurities and its characterization.

MATERIALS AND METHODS

The whole plant of Ipomoea leari was purchased in the month of July 2009 from Abirami Botanicals, Tuticorin, Tamil Nadu, India, and authenticated by Dr. D. Suresh Baburaj, Survey of Medicinal Plants and Collection Unit, Ootacamund, India. A specimen has been deposited at J.S.S College of Pharmacy herbarium, Ootacamund, India (Reference Voucher no-TIFAC 21).

Pharmacognostical studies and phyto-chemical studies

Quality of the dried leaves of the plant was evaluated by determining ash values and extractive values. These were performed as per Indian Pharmacopoeia (IP) 2007.[8] The ash values namely total ash, acid-insoluble ash, water-soluble ash and sulphated ash and extractive values were determined.

Fluorescence characters of the powdered plant

The drug powder was taken and treated with various chemical reagents such as sulphuric acid, hydrochloric acid, 5% iodine solution, 50% ferric chloride, 10% sodium hydroxide and ammonia solution, lead Acetate and the colour obtained was visualized under short UV light (254 nm) and long UV light (366 nm) in an UV chamber. The results are recorded in Table 1. TLC analysis of n-hexane, chloroform, ethyl acetate, and hydromethanolic extracts were carried out in various solvents according to the standard protocols.[91011]

Table 1

Fluorescence analysis of the powdered of Ipomoea leari

Extractive values

Extractive values were determined by using the shade dried parts of the selected plant materials powdering them and passing the powder through a sieve (No. 40).

Anatomical studies

Samples of different plant parts were cut; removed from the plant and fixed in FAA (formalin, 5 ml + acetic acid, 5 ml + 70% ethyl alcohol, 90 ml). The specimens were dehydrated after 24 hours of fixing with graded series of tertiary-butyl alcohol as per the schedule given by Sass.[12] Paraffin wax (melting point, 58–60°C) was added gradually until TBA solution attained super saturation to infiltrate the specimen. The specimens were then cast into paraffin blocks.

Photomicrographs

Microscopic descriptions of tissues were supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with a microscopic unit equipped with an Olympus Microscope, Model: BX41TF, Mode: E330-ADU1.2X (Digital camera), Olympus Corporation, Tokyo, Japan. For normal observations, bright field was used. For the study of crystals, starch grains and lignified cells, polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnifications of the figures were indicated by the scale-bars.[13]

Extraction procedure

The plant was chopped to small pieces and dried in shade. The dried Ipomoea leari root was powdered and passed through sieve No. 20 and extracted (600 g) successively with 4.8 L each of n-hexane (60–80°C), chloroform, ethyl acetate and hydromethanolic (1:1) solvents in a soxhlet extractor for 18–20 h. The extracts were concentrated to dryness under reduced pressure and controlled temperature (40–50°C) and then these Ipomoea leari extracts were henceforth called as ILH, ILC, ILE and ILHM, respectively.

Preliminary phytochemical screening of the extracts

The extracts were subjected to phytochemical tests to find the presence of major phytochemical constituents such as carbohydrates, proteins, amino acids, alkaloids, glycosides, saponins, sterols, flavonoids, phenolic compounds, fixed oil and fat, gum and mucilage, phytosterols and tannins according to standard methods.[141516]

Development of standard analytical parameters

Macroscopic evaluation, microscopic studies, physical parameters such as foreign matter, ash values, fluorescence analysis, extractive value and preliminary phytochemical analysis of various extracts of Ipomoea leari were performed according to the standard methods.[171819]

RESULTS

The macroscopic analysis revealed that Ipomoea leari is a twining perennial vine with hairy stems more than 7 m. Leaves were with blade, ovate in outline, 4–17 cm long, 3–16 cm wide, base heart-shaped, margins entire to deeply 3-lobed; leaf stalk 2–18 cm long.

Quality of raw materials

Ash values and extractive values

Ash values and extractive values were determined to assess the quality of collected raw materials. Results of the ash values and extractive values are given in Tables 2 and 3, respectively.

Table 2

Ash values of Ipomoea leari

Table 3

Extractive value of Ipomoea leari

Fluorescence analysis powdered plant

Fluorescence characters of the powder under UV light (254 and 366 nm) were determined and are tabulated in Table 1.

Toluidine blue, a polychromatic stain was used for staining the sections. The staining results were remarkably good. The dye rendered pink color to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage and blue to the protein bodies.

Microscopy of leaf

The leaf consists of prominent midrib and a lamina, both having dorsiventral symmetry [Figure 2a]. The lateral vein is also prominent [Figure 2b]. The lamina is smooth and shows dorsiventral differentiation of the mesophyll [Figure 2c]. The leaf is 100–120 µm thick. The adaxial epidermis consists of a wide square shaped thin walled cuticle. The epidermis is 25 µm thick. The thin abaxial epidermis comprises narrow cylindrical cells. The adaxial epidermis is stomatiforous. The mesophyll includes adaxial zone of two layers of short vertically elongated columnar palisade cells and six or seven layers of lobed loosely arranged spongy parenchyma cells. The veinlet is prominent having conical collateral vascular strand and parenchymatous bundle sheath with adaxial extension [Figure 2c].

Figure 2

Microscopy of ipomoea leari leaf. (a) T.S. leaf - through midrib, (b) T.S. leaf - through lateral vein, (c) T.S. Lamina, (d) T.S. Midrib enlarged, (e) vascular tissues on midrib - enlarged, (f) abaxial epidermis showing, stomata, (g) adaxial epidermis showing basal cell of trichome, (h) veination pattern of leaf through vein islet, (i) vein termination enlarge, (j) epidermal non glandular trichome, (k) basal cell of the trichom, (l) lactifer in the leaf, (m) T.S. distal part of the petiole, (n) T.S. proximal part of the petiole, (o) proximal part of petiole enlarged view, (p) T.S. stem entire view

The midrib consists of an adaxial thick, short cone or a hump and a wide and thick semicircular abaxial midrib which is 800 µm thick. The adaxial hump is 300 µm wide and the abaxial part is 1.1 mm wide. The midrib consists of a thin epidermal layer of small cells. Within the adaxial hump occurs a group of compact collenchyma cells. In the abaxial part, the ground tissue is homogeneous with angular compact parenchyma cells.

The vascular strand is thick and wide. It is 700 µm wide and 200 µm thick. The vascular bundle is bicollateral having isolated small groups of phloem elements distributed all along the entire width of the xylem strand [Figure 2d and e].

The abaxial epidermal cells, as seen in surface view, are wide with thin wavy anticlinal walls. The epidermis has stomata. The stomata are broadly elliptical measuring 20 × 25 µm in size. The stomata are either actinocytic with radiating subsidiary cells or diacytic with two subsidiary cells lying at right angles both guard cells [Figure 2f].

The adaxial epidermis is apostomatic. The epidermal cells are fairly thick walled and the walls are straight or slightly wavy. The epidermal cells from which the trichomes arise are circular and they are surrounded by radially elongated rosette of cells [Figure 2g and K].

The veins are thin, less prominent and straight. The veins consist of one or two rows of vascular elements. The vein-islets are wide with distinct vein borders [Figure 2h]. These islets are polygonal in outline. The vein-terminations are prominent. They are simple or more frequently branched to form a dendroid appearance; the terminal part of the termination bears dilated tracheids [Figure 2i and Figure 3Dd].

Figure 3

Microscopy of ipomoea leari stem. (q) T.S. of stem a sector, (r) T.S. of stem corticle portion, (s) T.S. of stem outer phloem, (t) T.S. of stem inner phloem, (u) laticifers in the pith, (v) T.S. of root entire view, (w) a sectar enlarged, (x) cortical cells of root showing starch grains and crystals, (y) glandular trichomes on the lamina, (z) one gland enlarged, (Aa) xylem elements in the powder, (Bb) fibre and vessels in the powder, (Cc) tracheids in the powder, (Dd) one vessel element, (Ee) a tracheid, (Ff) pits of the tracheids enlarged

Nonglandular epidermal trichomes come along the veins and the leaf-margins [Figure 2j]. The trichomes are unicellular, unbranched, thin walled and broad at the base, tapering towards the tip. The trichomes arise from a wide circular epidermal cell surrounded by radiating basal cells [Figure 2k]. The trichomes are 210–320 µm long. Narrow, long canal like laticifers are wide spread in the leaf mostly associated with the veins. The laticifers are non articulated and non - anastamosing (non septate and unbranched) type. No prominent cell inclusions are evident in the laticifer. The canal is 15 µm thick [Figure 2l].

The distal and proximal parts of the petiole were studied. The distal part of the petiole is circular with narrow adaxial groove [Figure 2m]. It is 1.8 mm thick. Proximal part of the petiole is slightly wider and the adaxial groove is wide and shallow [Figure 2n]. It is 2.4 mm in horizontal plane and 1.7 mm in vertical plane. The structure of the proximal and distal parts of the petiole are similar. They have a thin layer of circular epidermal cells. The vascular system includes a wide bowl shaped abaxial strand and two small elliptical adaxial bundles [Figure 2o]. The vascular bundles are bicollateral: They have short, parallel lines of circular thick walled xylem elements and small nests of phloem elements located on the inner and outer sides of the xylem strands [Figure 2o].

Microscopy of stem

The stem is circular in cross sectional view. It is 2.3 mm thick and consists of an epidermal layer of unicellular cells, wide cortex, thin hollow vascular cylinder and wide pith [Figures 2p and 3q]. The epidermal cells are 25 µm thick, vertically rectangular and have thick cuticle. The cortical cells are homogenous and parenchymatous. The cells are angular, thick walled and compact [Figure 3r and Cc]. The Vascular cylinder comprises closely aggregated short radial files of primary xylem elements. The elements are wide angular and thin walled. Phloem occurs on both outer and inner sides. The phloem elements are in small nests and scattered near the xylem elements [Figure 3s and t]. The xylem elements are 40 µm wide. The pith cells are circular, thin walled and parenchymatous. Some of the pith cells are smaller and thick walled and they are laticifers [Figure 3u].

Microscopy of root

The root sample measuring 1.6 mm thick was studied. It consists of uniformly thick and continuous periderm, wide cortex and thick solid vascular cylinder [Figure 3v and w]. The periderm is 150 µm thick. It includes thin walled homogenous phellem cells which occur in regular radial rows. The cell cortex is 350 µm wide; it is parenchymatous and the cells are compact and thin walled. Starch grains and calcium oxalate crystals are abundant in the cortical cells [Figure 3x]. The vascular cylinder is 700 µm in diameter. It consists of a thin layer of endodermis (endodermoid layer), tetrarch primary xylem strands and four separate masses of secondary xylem. There are four small primary phloem strands alternating with primary xylem. The secondary xylem segments include wide thin walled vessels and xylem fibres; secondary phloem is found as a thin layer outside the secondary xylem units [Figure 3w].

Powder microscopy

Fragments of lamina possessing subsessile, spherical glandular trichomes in surface view [Figure 3y]. Isolated glands are also seen in the powder [Figure 3z]. The glands are capitate type. They are located in shallow pits on the lamina. The gland has a basal cell of the epidermis, a short stalk cell and 4 celled or many celled spherical body [Figure 3y and z]. The glands are 50–180 µm in diameter. Xylem elements including fibres, tracheid vessel elements and parenchyma cells are common in the powder [Figure 3Aa-Cc]. The fibres are libriform type, long and narrow with tapering ends and lignified walls. No pits are evident on their walls [Figure 3Bb]. The fibres are up to 850 µm long and 20 µm wide. Vessel elements are short, wide and cylindrical [Figure 3Bb and Dd]. They have dense lateral wall pits which are horizontally elongated, multiseriate and olioserate [Figure 3Dd]. The end wall perforation is wide, simple and horizontal in orientation. The elements are 150–250µm long.

Tracheids [Figure 3Cc-Ee] are equally abundant as the fibres. The tracheids appear similar to the fibres in shape and size. The tracheids are long, narrow and needle shaped. The tracheids possess dense size measure 300–550 µm in length, well developed bordered pits on the lateral walls [Figure 3Ee and Ff]. The pits are uniseriate or multiseriate. Parenchyma cells are widespread in the powder. They are mostly cuboidal to rectangular in shape and have thin walls and wide lumen. They are found in small groups or in isolated cells. No cell inclusions are evident in the cells.

Nature of the extracts and yield

The ILH yielded yellowish green sticky semisolid (2.37 g), ILC yielded green semisolid (6.07 g), ILE yielded dark blackish green solid (6.73 g) and ILHM yielded brownish solid (21.54 g), extracts respectively. All the extracts were stored in a refrigerator at 4°C until further use.[11]

Preliminary phytochemical studies

The results of the preliminary phytochemical studies are given in Table 4. The data reveal the presence of carbohydrates, flavonoids, glycosides, steroids and phenols in ILH, ILC and ILE. But ILHM shows presence of carbohydrates, flavonoids, saponins, and phenols. Alkaloids and terpenoides are not present in any of the extracts.

Table 4

Phytochemical screening of Ipomoea leari extracts and fraction

DISCUSSION

Standardization of herbal medicines is necessary to ensure that high quality herbal materials are available in the market. This analysis is designed to help ensure the identity, quality and purity of herbal drugs. The standardization of crude drug is an integral part of establishing its correct identity as per herbal pharmacopoeia. The present study is a step in this direction. Ethnobotanically, we found that tribal people used Ipomoea leari (whole plant) for various applications such as antimicrobial, analgesic, spasmogenic, hypoglycemic, hypotensive, anticoagulant, anti-inflammatory, psychotomimetic and anticancer activities without standardization of source drug. Anatomical studies of the leaves, stem and root were performed to identify the special micoscopical characters of plant.[2021]

Microscopically, the features indicate that the leaf consists of prominent midrib and the lamina. The lamina is smooth and shows dorsiventral differentiation of the mesophyll. The mesophyll includes adaxial zone of two layers of short vertically elongated columnar palisade cells and six or seven layers of lobed loosely arranged spongy parenchyma cells.

The veinlet is prominent having conical collateral vascular strand and parenchymatous bundle sheath with adaxial extension. The stomata are either actinocytic with radiating subsidiary cells or diacytic with two subsidiary cells lying at right angles both guard cells. Nonglandular epidermal trichomes come along the veins and the leaf-margins. The trichomes are unicellular, unbranched, thin walled and broad at the base, tapering towards the tip.

Stem microscopically consists of an epidermal layer of unicellular cells wide cortex, thin hollow vascular cylinder and wide pith. In that the vascular cylinder comprises closely aggregated short radial files of primary xylem and the pith cells are circular, thin walled and parenchymatous. Some of the pith cells are smaller and thick walled and they are laticifers. Microscopy indicates that the root consists of uniformly thick and continuous periderm, wide cortex and thick solid vascular cylinder. Root shows the presence of starch grains and calcium oxalated crystals are abundant in the cortical cells. The results obtained will helpful for further standardization of this plant and in future these characters may be compared with new batches of the same plant material. Adulterants and substituents can also be detected.[22]

Ipomoea leari, percentage extractives and ash analysis were carried out. It was found that plant is of good quality as seen from their ash values and extractive values. Ash values of a drug give an idea about the impurities present along with drug. Extractive values are useful for the determination of exhausted and adulterated drugs. Extractive values are also useful to evaluate the type of chemical constituents present in the crude plant.[2324] Results of the fluorescence analysis reveled that the powder material showed blue colour for powdered drug in near UV 254 nm, reddish blue under far UV light 366 nm when treated with acetic acid. Pale green colour when treated with hydrochloric acid was observed under near UV 254 nm while yellowish green colour was observed for the powder drug under far UV light 366 nm. Reddish-orange colour when treated with 50% ferric chloride was observed under near UV 254 nm while reddish-orange colour for powder drug under far UV 366 nm. This procedure is a simple preliminary pre-requisite for a detailed photochemical investigation. Various tests have been conducted qualitatively to find out the presence or absence of bioactive compounds.[25] Different chemical compounds such as saponins, flavonoids, phenols, glycoside and sugar were found. This analysis suggests that, Ipomoea leari probably contains active agent(s) and this provides the basis for the traditional use of this plant in many human alimentary disorders.

CONCLUSION

The whole plant Ipomoea leari, was evaluated for its quality by performing ash values and extractive values. Anatomical studies of the leaf, root and stem were performed to identify the special microscopic characters. The findings suggest that the microscopic features and the numerical standards reported in this work may prove useful for the compilation of a suitable monograph for the proper identification and authenticity of the Ipomoea leari plant.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.