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ORIGINAL ARTICLE
Year : 2018 | Volume
: 37
| Issue : 3 | Page : 120-126
Detailed pharmacognostical and analytical profile of telosma pallida (l.) kurz. (leaf): A folklore medicinal plant of Gujarat State
Riddhi Dineshkumar Kanakhara1, Harisha Channappa Rudrappa1, Vinay Janardan Shukla2, Rabinarayan Acharya3
1 Pharmacognosy Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurveda University, Jamnagar, Gujarat, India
2 Analytical Chemistry Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurveda University, Jamnagar, Gujarat, India
3 Department of Dravyaguna, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurveda University, Jamnagar, Gujarat, India
Correspondence Address:
Dr. Riddhi Dineshkumar Kanakhara
8th Digvijay Plot, “Shree Satyanarayan Krupa”, Nr. Deepak Drug House, Jamnagar - 361 005, Gujarat
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/asl.ASL_97_17
Background: Telosma pallida (L.) Kurz., is one of the traditional medicinal plants from Asclepiadaceae family. It is abundantly available in Gujarat, India during monsoon. It is a slender and deciduous climber used traditionally in the treatment of various diseases such as cough, cold, skin diseases etc., Aims: To study the leaves of Telosma pallida (L.) Kurz. for detailed Pharmacognostic characters and Analytical Profile. Materials and Methods: In this work pharmacognostic, preliminary phytochemical screening, fluorescence analysis, and HPTLC profile of leaves were determined following standard procedures. Results: The leaves are simple, entire, opposite, cordate in shape, the upper surface is dark green and lower surface is light green in colour. The Transverse section of a leaf through petiole shows the presence of uniseriate multicellular warty trichomes and laticifer cells at the adjacent side of the phloem. Surface study shows paracytic and anomocytic type of stomata. Preliminary physicochemical analysis shows water soluble extractive value as 28.44 ± 2.89% w/w and alcohol soluble extractive value as 14.60 ± 0.79% w/w. Preliminary phytochemical analysis of extracts shows the presence of glycosides, alkaloids, carbohydrates, proteins, amino acids and flavonoids. Conclusion: The information obtained from this research work may be useful to establish the botanical as well as analytical standards for the leaves of T. pallida.
Keywords: Ayurveda, folklore, pharmacognosy, surface study, Telosma pallida
World Health Organization (1978) report shows that 80% population of the world depends on traditional herbal medicine for their primary healthcare.[1] Plants are the reservoirs of naturally occurring chemical compounds and of structurally diverse bioactive molecules. The extraction of bioactive complexes from the plants and their quantitative and qualitative evaluation is important for exploration of new biomolecules and their activities. Telosma pallida (L.) Kurz., belonging to Milkweed family (Asclepiadaceae) is one of the useful traditional medicinal plants of Gujarat.[2] In Guajarati it is well known as “Radarudi” or “Vachhanti”.[3],[4] Traditionally, T. pallida is used in the treatment of various diseases such as a cough, cold, skin diseases and so on. Further the leaves and flowers of this plant are commonly used as a vegetable. In-vitro anti-inflammatory and anti-arthritic potential of the T. pallida leaf extract have been reported.[5] Research data regarding detail pharmacognostic and analytical evaluation of T. pallida is not available till date. The present research work is focused on ascertaining by Pharmacognostic characters and analytical parameters of T. pallida leaf.
Materials and MethodsCollection and authentication of plant drug
The sample drugwas collected with the help of local farmer, from its natural habitat of Jamnagar in the outskirts of Gujarat state during monsoon season and further authenticated at I.P.G.T & R.A, Pharmacognosy Lab. The plant was washed with tap water, dried and then Herbarium sheet (No. Phm-6172/15) was submitted to Pharmacognosy Lab., I.P.G.T & R.A. for future reference. Healthy and unaffected leaves were stored in FAA solution (Formalin: Acetic acid: 70% Ethyl alcohol) in the ratio of 5:5:90 ml.[6]
Powder preparation
The remaining parts of healthy leaves were subjected to shade drying and were subsequently powdered out by mechanical mixture and passed through 80# sieve. The powdered leaves were packed in an airtight glass container and used for organoleptic, microscopy, physicochemical and fluorescence analysis studies.[6],[7]
Extract preparation
Exactly weighted 5g of leaves powder was macerated with 100ml solvent (Water, chloroform, and methanol) in a closed conical flask for 24 hours. Thereafter, samples were filtered with simple filter paper and extract was collected. These extracts were used for preliminary phytochemical test. The methanol soluble extract was used for HPTLC profile.
Pharmacognostic investigation
Morphological features
The morphological features of the fresh leaves such as Phyllotaxy, venation, size of lamina and petiole; shape, margin, apex/tip were studied. Organoleptic characters of fresh leaves were observed.[6],[7]
Microscopy
The sections for microscopy were prepared by free hand. The section of the petiole and lamina through midrib was observed under Carl Zeiss microscope. Some thin sections were selected and observed in distilled water then stained with phloroglucinol along with hydrochloric acid and again examined to assess different cellular structure and lignified elements.[6],[8],[9]
Leaf surface study
Leaf pieces (Upper & lower surface) were peeled off mechanically and kept on slide to observe type of stomata, stomatal index (SI), and measurement of stomata under Carl Zeiss microscope. The middle part (midrib) of the leaf was observed under microscope with distilled water to calculate Palisade ratios.[6],[7]
Stomatal Index is obtained by the following formula:
All the readings were taken in triplicate and the results were tabulated as mean ± standard error mean (SEM).
Powder microscopy
For powder microscopy, slides were prepared by using water, chloral hydrate as a clearing agent. Leaves powder slides were also prepared for histochemical and micrometric evaluation.[6]
Organoleptic characters of the powder
Organoleptic characters such as colour, odour, taste, and feel of drug to touch were noted down by sensory observations.[8]
Micrometric evaluation
Measurement of the length, width of different cell layers along with their contents of T.S and powder characters were taken into consideration for micrometric evaluation.[8]
Histochemical studies
Transverse section, as well as powder of leaves were subjected to histochemical evaluation by stain with phloroglucinol and concentrated hydrochloric acid (HCl) for lignified tissues, iodine solution for starch grains, 5% ferric chloride (5% FeCl3) for tannin and glycerine for mount.[9],[10]
Physico-phytochemical evaluation
Physico-chemical parameters such as Loss on Drying (LOD), Ash value, extractive value, pH (5% w/v solution in water at room temperature) were determined. Phytochemical screening of the leaves powder was carried out for Alkaloid, Carbohydrate, and Proteins etc., as per standard procedures.[11],[12],[13] All the readings were taken in triplicate and the results were tabulated as mean ± standard error of mean (SEM).
High-performance thin layer chromatography profile
High-Performance Thin Layer Chromatography (HPTLC) profile of the methanol extract of leaves powder was carried out as per standard procedures.[11]
Fluorescence analysis
The fluorescence analysis of the powdered of leaves of T. pallida was carried out using various solvents and observed under Ultra-Violet (UV) light.[14],[15]
Observations and ResultsMorphological characteristics of the leaf
Leaves were simple, opposite, petiole 3-5 cm long and 0.1-0.3 cm broad; leaf blade was ovate to cordate in shape, simple entire margin, apex was acute to acuminate with a subcordate base. Leaves were of various sizes i.e. 6-20 cm length and 3.5-10.5 cm width. Venations were reticulate and 5-6 pairs of lateral veins were prominently raised on dorsal side. The upper surface of leaves was dark green in colour while lower surface was light green in colour. Leaves were astringent in taste and had a characteristic odour [Figure 1].
Figure 1: (a) Natural Habitat, (b) measurement of leaf, (c) T.S of petiole, (d) epidermal cell with warty trichome, (e) vascular bundle and meristeal, (f) group of starch grainMicroscopy
Transverse section of leaf petiole
Diagrammatic section of petiole shows a circular to orbicular shaped structure followed by ground tissue and centrally located vascular bundle.
Detailed transverse section of petiole shows single layered epidermis with cuticle. Some of the epidermal cells are interrupted with multicellular warty trichomes. Three to four layers of compactly arranged collenchyma cells form the hypodermis. The major part of the section comprises of ground tissue made up of parenchymatous cells embedded with prismatic crystals of calcium oxalate, oil globules, and simple starch grains. Vascular bundle is centrally located open and collateral; phloem is present at the lower region of the xylem made up of phloem fibres and sieve elements. Some of the laticiferous cells are also observed at adjacent side of the phloem. Xylem is made up of xylem parenchyma and its fibres. Two meristeles are located at the edges of main vascular bundle [Table 1] and [Figure 1].
Table 1: Micrometric measurements of T.S. of petiole of Telosma pallida leafTransverse section of leaf lamina through midrib
Leaf is dorsiventral type and differentiated into upper palisade parenchyma and lower mesophyll tissue. Epidermis is single layered with both upper and lower epidermis interrupted by stomata. Transverse section through midrib shows large vascular bundle present at the centre.
Epidermis
Upper epidermis consists of single layered barrel shaped compactly arranged cells. Some of the cells lead to form multicellular warty trichomes. The lower epidermis also consists of single layered epidermal cells as in the upper epidermis. Both the epidermises are covered with thick cuticle.
Mesophyll
Mesophyll is differentiated into upper single layer palisade cell and lower spongy parenchymatous layers. Cells are rarely loaded with oil globules and prismatic crystal of calcium oxalate. There are 5-7 layered loosely arranged rounded to oval shaped spongy parenchyma with many air chambers located in the lower epidermis.
Vascular bundle
Vascular bundle covered by single layered bundle sheath is situated at the centre. Xylem is towards upper epidermis while, radially arranged metaxylem is toward the lower epidermis and protoxylem towards the upper epidermis. Phloem is situated towards lower epidermis. Laticifer cells are observed at the region of the phloem. Rest of the central part of the section is made up of parenchyma cells. Three to five layered collenchyma cells are present beneath the both surfaces which give mechanical support to the vascular bundle [Table 2] and [Figure 2].
Figure 2: (a) T.S of leaf through midrib, (b) lignified vascular bundle, (c) length of palisade cell, (d) width of palisade cell, (e) paracytic stomata, (f) anomocytic StomataSurface study
Both the upper and lower surfaces of the leaf were peeled out and observed under microscope; both the epidermises show epidermal cells, wavy parenchyma, oil globules, Paracytic stomata and anomocytic stomata [Table 3] and [Figure 2].
Powder microscopy
Organoleptic characters
Organoleptic characters of leaves powder were carried out and the results are depicted in the [Table 4].
Micrometric evaluation of leaves powder
Diagnostic characters of leaves powder of T. pallida show oil globules, prismatic, rosette and cluster crystals of calcium oxalate, fragment of stomata, epidermal cells, multicellular warty trichomes, annular vessels and its Micrometric measurements are depicted in the [Table 5] [Figure 3].
Figure 3: (a) Leaf powder, (b) epidermal cell with stomata, (c) cluster crystal, (d) multicellular warty trichomes, (e) prismatic crystal, (f) annular vessels, (g) border pitted vessels, (h) lignified fibresHistochemical evaluation
T.S. of leaf and its powder shows presence of starch grains, calcium oxalate crystals and lignified cells through histochemical tests and results are depicted in [Table 6].
Physico-chemical analysis of leaves Powder:
The results of physicochemical evaluation of leaf powder are depicted in [Table 7].
Preliminary phytochemical evaluation of leaves powder
The preliminary phytochemical screening was carried out on water, methanol and chloroform extracts to find out the presence or absence of primary and secondary metabolites [Table 8].
Table 8: Preliminary phytochemical evaluation of Telosma pallida leaves powderChromatographic analysis (high-performance thin layer chromatography)
Chromatographic investigation was carried out as per the standard materials and methods. The solvent system which was designed for HPTLC studies was in the ratio Toluene: Ethyl acetate: Acetic acid (7: 2: 1 v/v). Results are depicted in [Table 9] [Figure 4].
Table 9: High-performance thin layer chromatography studies of methanolic extracts at 254 nm-366 nm
Figure 4: (a) Plate at 254 nm, 366 nm and after spray, (b) three dimensional graph at multi-wavelength, (c) peaks at 254 nm, (d) three dimensional graph at 254 nm, (e) peaks at 366 nm, (f) three dimensional graph at 366 nmSample loading: Methanolic extract of Leaf (1 mg/ml)Spray reagent: Vanillin – Sulphuric acid.Fluorescence analysis of leaves powder
The powder was treated with different solvent and changes in colour were examined under daylight and Ultraviolet light (365 nm). The results of the observation are depicted in [Table 10].
DiscussionPlant morphology showed leaves opposite, broad at the base and acute to acuminate at the apex, entire margin. The colour of leaves showed pale green upper and light green in lower surface. Five to six pairs of lateral veins were prominently raised on dorsal side.
Transverse section of petiole showed single layered epidermis interrupted by Multicellular warty trichomes. Ground tissue made up of parenchyma cells loaded with prismatic crystals of calcium oxalate, oil globules, and simple starch grains. Vascular bundle was centrally located and was open and collateral. Transverse section of the lamina through the midrib showed upper and lower epidermis interrupted by stomata, prismatic crystal of calcium oxalate and uniseriate multicellular warty trichomes. Chloroplast and oil globules were present in upper palisade and lower spongy parenchyma.
Stomatal index of the leaves showed 25.37 ± 1.08, 29.67 ± 0.63 in upper and lower epidermis respectively, palisade ratio is 1/3. These values are constant even in different environmental conditions, so they play an important role in the identification of plant even up to the species level. Histochemical evaluation of leaves revealed presence of lignified cells, oil and tannin contents in T.S as well as powder.
Powder microscopy of leaves showed diagnostic characters such as multicellular warty trichomes, starch grains, fragment of stomata, epidermal cells, rosette, prismatic and cluster crystals of calcium oxalate.
Physico-chemical parameters of leaves such as Ash value and Acid insoluble ash were 14.46 ± 0.30% w/w and 0.26 ± 0.06% respectively, these values indicate the presence of inorganic and salt materials.[16] pH value of the powder showed 5.42 which is a weak acid, so it may be comparatively rich in organic acids. LOD is designed to measure the amount of water and volatile matters in a sample. Its value is 14.96 ± 0.13% w/w.[16] Water soluble extractives were higher (28.44% w/w) which indicates that the drug can be used as Kaṣāya, hima, phāṇṭa preparations.[17] Alcohol soluble extractive shows 14.60 ± 0.79% w/w which indicates the presence of polar constituents.
Preliminary Phytochemical screening showed alkaloids, Tannins and phenols present in water and alcohol soluble extracts. Saponins, carbohydrates, glycosides were present in water soluble extract whereas steroids were present in chloroform extract. These phytochemicals are medicinally active as well as exhibit physiological activity.
HPTLC profile showed nine spots at 254 nm and six spots at 366nm, in which five Rf value (0.03, 0.61, 0.65, 0.86, 0.97) are common in both. After spray with Vanillin Sulphuric acid, four spots were observed among which, spot at 0.65 Rf was found to be common at 254 nm and 366 nm respectively.
Fluorescence is the phenomenon exhibited both in visible and UV- light by various chemical components present in the plant material. Some plant drugs are often evaluated qualitatively in this way and this can be used as effective tool to identifying and authenticating original plant material.
ConclusionLeaf morphology and its constants such as Surface study, Anatomy of Petiole and midrib are useful for identification and standardization of this drug. The physicochemical parameter, HPTLC profile and Fluorescence analysis of the leaves can be used for ensuring quality and purity of the raw drug. The information obtained from this research may be useful to establish certain botanical standards.
Financial support and sponsorship
IPGT and RA, Gujarat Ayurved University, Jamnagar, Gujarat, India.
Conflicts of interest
There are no conflicts of interest.
References
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