|Year : 2017 | Volume
| Issue : 1 | Page : 26-33
Pharmacognostic and physicochemical evaluation of homoeopathic drug: Erigeron canadensis L.
Padma Rao Pogaku, Subramanian Palani, Sudhakar Penthala, P Sreeman Narayana, Nagaraju Mukkamula
Drug Standardisation Unit, Hyderabad, Telangana, India
|Date of Web Publication||23-Feb-2017|
Drug Standardisation Unit, O.U.B. 32, Road No. 4, Habsiguda, Hyderabad - 500 007, Telangana
Source of Support: None, Conflict of Interest: None
Background: Erigeron canadensis L. is an erect annual herb belonging to the family Asteraceae. Aerial parts are used in Homoeopathy for bruises, cough, dysuria, gonorrhea, haemorrhages, haemorrhoids, spermatorrhea, and wounds. Objective: The pharmacognostic and physicochemical studies have been carried out to facilitate the use of correct species and lay down standards of raw drug materials. Materials and Methods: Pharmacognostic studies of the leaf and stem of authentic samples of E. canadensis L. have been carried out. Physicochemical parameters of the raw drug include extractive values, ash value, and formulation; besides weight per mL, total solids, and alcohol content, high-performance thin layer chromatography (HPTLC) and ultraviolet (UV) studies are given. Results: Epidermal cells often possess crystals of calcium oxalate. Stomata are anomocytic, anisocytic, and tetracytic types. Trichomes are uniseriate and conical in structure. The mid vein in transection is flat on adaxial and is ribbed toward abaxial, with a secretory cavity beneath the central vascular bundle. Stem in transection is round. The vascular tissue is made of several vascular bundles in a ring. Crystals of calcium oxalate occur in the epidermis, cortex, and pith of stem. In mature stem, secondary xylem is well developed with a reduced phloem. The determined physicochemical data, namely, extractive values, ash values, and preparation of for raw drug and weight per mL, total solids, and alcohol content besides UV and HPTLC profile for finished product are provided. Conclusions: The presented morphoanatomical features along with powder microscopic and organoleptic characters and physicochemical data are diagnostic to establish the standards for ensuring quality and purity of the drug.
Keywords: Erigeron canadensis, High-performance thin layer chromatography, Homoeopathy, Pharmacognosy, Physicochemical, Secretory cavities
|How to cite this article:|
Pogaku PR, Palani S, Penthala S, Narayana P S, Mukkamula N. Pharmacognostic and physicochemical evaluation of homoeopathic drug: Erigeron canadensis L. Indian J Res Homoeopathy 2017;11:26-33
|How to cite this URL:|
Pogaku PR, Palani S, Penthala S, Narayana P S, Mukkamula N. Pharmacognostic and physicochemical evaluation of homoeopathic drug: Erigeron canadensis L. Indian J Res Homoeopathy [serial online] 2017 [cited 2017 Aug 20];11:26-33. Available from: http://www.ijrh.org/text.asp?2017/11/1/26/200841
| Introduction|| |
Erigeron canadensis L. (Syn. Conyz a canadensi s (L.) Cronq.), popularly known as “Canada fleabane” in English, is a leafy annual herb belonging to the family Asteraceae. It is distributed in the European part of the former USSR, the Caucasus Western and Eastern Siberia, Central Asia, Asia Minor, Iran, Japan, China, and North America. In India, it is found growing in Western Himalayas, Punjab, Upper Gangetic plains, Valleys of Kashmir, Shillong, Western Ghats, and Nilgiris. The tincture of fresh aerial parts is used as medicine in Homoeopathy. It is used in the treatment of black eye, bruises, cough, dysuria, gonorrhea, haematocele, haemorrhages, haemorrhoids, spermatorrhea, and wounds. It has been proved in Homoeopathy by W.H. Burt as given in American Homoeopathic Observer, 1966. Wilmot Moore has given it with success in cases of placenta previa. Development and utility of advanced analytical techniques are indispensable in drug research, for the identification of active compounds and characterization of chemical structure, quantification, and laying down standards for scientific quality control. To solve complex natural matrix, it is mandatory to use combination of advanced techniques. Further, it is also helpful to analyze multiple compounds or multiple classes of components. Advanced analytical techniques (high-performance thin layer chromatography [HPTLC], liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, and nuclear magnetic resonance) are needed for better understanding and to ensure the product credibility.
Chemically, the plant is reported to contain erigeron oil which acts like turpentine but is less irritant and stimulating. The oil consists of d-limonene and terpineol, besides solid dihydrochloride, camphene, and germacrene D [Figure 1]. Sphingolipids and its derivatives (β-D-glucopyranoside) were isolated from ethyl acetate fraction along with β-sitosterol, stigmasterol, β-sitosterol-3-O-β-D-glucoside, and harmine. About fifty components were identified from the oil, the important being limonene and trans-alpha-bergamotene. Five compounds were isolated from ethanol extract and identified as scutellarin, luteolin-7-O-beta-D-glucuronide, quercetin, quercetin-3-O-β-D-glucopyranoside, and luteolin. Twelve flavonoids were isolated from ethanolic extract of the whole plant.
A review of literature reveals no pharmacognostic standards laid down for this drug, except some review works on trichome diversity  and organoleptic distribution of calcium oxalate crystals in Conyza spp. In view of the significance and importance of the drug, pharmacognostic and physicochemical standardization studies were carried out to lay down specific standards in homoeopathic perspective.
| Materials and Methods|| |
The plant material E. canadensis was supplied by the Survey of Medicinal Plants and Collection Unit, Nilgiris, Tamil Nadu. The leaves and stems were fixed in formaldehyde-acetic acid-alcohol, dehydrated through xylene-alcohol series, and embedded in paraffin wax. The sections cut between 8 and 10 μm were stained with crystal violet and basic fuchsin combination as per the Johansen method. Epidermal peels were obtained by gently scraping and peeling with a razor blade. Then, the peels were stained in safranin and mounted in glycerin. The photomicrography was done on BX-53 trinocular microscope attached with a digital Camera.
The air-dried whole plant was coarsely powdered to 10/44 (sieve size) and subjected to determination of loss on drying at 105°C, total ash, water soluble ash, acid insoluble ash, extractability in different solvents, physicochemical constants, and ultraviolet (UV) aspects of mother tincture, following official methods. Mother tincture was prepared as per the Homœopathic Pharmacopœia of the United States Convention  by percolation method. One hundred grams of coarse powder of the drug was suspended in 677 mL of 95% alcohol and 350 mL of purified water for 24 h at room temperature. It was filtered and made up to 1000 mL using the same solvent ratio.,
High-performance thin layer chromatography analysis
25 mL mother tincture was evaporated on water bath to remove alcohol. The residue was extracted thrice with 25 mL chloroform. Concentrated chloroform extract was used for the HPTLC study. The concentrated chloroform extract was spotted in the form of bands of length 6 mm with 100 μL syringe on precoated silica gel aluminum plate 60 F254, (5 cm × 10 cm with 0.25 mm thickness; Merck, Darmstadt, Germany) using a Linomat-V sample applicator (Camag, Muttenz, Switzerland). A constant application rate of 6 μL/s was employed. The space between the bands was 10 mm. The slit dimension was kept at 5 mm × 0.45 mm and scanning speed at 20 mm/s was employed. The mobile phase of chloroform: methanol (9:1 v/v) and 10 mL was used for chromatography. Linear ascending development was carried out in a 10 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase at room temperature for 20 min. The length of the chromatogram run was 8.5 cm, and subsequent to the development, the thin layer chromatography (TLC) plates were dried in a current of air with the help of hot air dryer in a wooden chamber with adequate ventilation. Densitometric scanning was performed (Camag TLC scanner III) at 254 nm and 366 nm by reflectance scanning and operated by winC ATS software (v 1.4.3 Camag Muttenz, Switzerland).,,
| Observations and Results|| |
The leaves are 2.5–4 cm long, sessile, linear-obovate with rough surface. Stems are 2–5 mm thick, ridged with coarse surface and densely hairy.
In surface, epidermal cells of adaxial are polygonal anisodiametric to linear, sides thin, curved to wavy and sinuate, surface smooth and those of abaxial are similar with deeply sinuate sides, contents scanty with sandy crystals of calcium oxalate occurring frequently. Epidermal cells are more frequent on adaxial, 2430/mm 2 and lesser toward abaxial, 1440/mm 2; stomata present on either sides of anomocytic, anisocytic, and tetracytic types. Stomatal number was 150/mm 2 for adaxial and 162/mm 2 for abaxial; stomatal index was 5.82 for adaxial and 6.2 for abaxial. Trichomes are uniseriate macroform conical hair, occurring common, more on veins [Figure 2]a and [Figure 2]b. In transection, leaf midvein is flat on adaxial and ridged prominently on abaxial, 302–432 μm (376) in thickness, covered by few conical hairs. Lamina is undulated often with elevated trichome bases, 108–194 μm (150) thick. Epidermis is single-layered, cells tabular and barrel-shaped, few oval to spherical, larger as trichome bases, cuticle slightly thick, contents scanty, with acicular crystals of calcium oxalate in few. Epidermal cells lie over the midvein oval to spherical, tabular and barrel-shaped, larger on abaxial. Stomata occur on both sides with guard cells raised above the surface. Mesophyll is dorsiventral, palisade two-layered on adaxial, cells cylindrical, columnar, 22–38 μm long and 8–19 μm (14) wide, filled with chloroplasts. Spongy parenchyma cells are polygonal, spherical, and cylindrical with few intercellular spaces, contents dense with chloroplasts [Figure 2]a and [Figure 2]b. Ground tissues at midvein are with two-layered collenchyma on adaxial side and single-layered on abaxial, cells angularly thickened. Parenchyma is 2–3 layered on adaxial and 4–6 layered on abaxial contents in few with chloroplasts. A secretory canal enclosed by an epithelial layer is conspicuous beneath the central vascular bundle, 16–50 μm (29) in diameter [Figure 3]a. Secretory canals also occur attached to veinular bundles of lamina [Figure 3]b. Midvein vascular bundle is arcuate, endarch, collateral, conjoint, 112–134 μm (125) in diameter. The tracheids/vessels arranged in radial rows, and in longitudinal section, they show helical or scalariform thickenings and rarely annular. The phloem is toward abaxial with phloem parenchyma, blast fibers, and sieve elements associated with companion cells. The margin is rounded [Figure 3]a.
|Figure 2: (a) Epidermal cells of leaf adaxial surface X 416, (b) epidermal cells of leaf abaxial surface X 625. Uch: Uniseriate conical hair; am: Anomocytic stomata; ts: Tetracytic stomata; an: Anisocytic stomata; cr: crystals|
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|Figure 3: (a) Transection of leaf at midvein X 162, (b) Transection of leaf lamina X 111, (c) Transection of young stem X 45. Ade: adaxial epidermis; co: Collenchyma; pl: Palisade; sp: Spongy tissue; p: Parenchyma; sc: Secretory cavity; vb: Vascular bundle; abe: Abaxial epidermis; h: Hair; st: Stomata; e: Epidermis; scl: Sclerenchyma; ph: Phloem; x: Xylem; pi: Pith|
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In transection, it is rounded with ridges and furrows covered by uniseriate hairs. Epidermis is single-layered, cells tabular and barrel-shaped, polygonal to spherical, and larger over ridges, walls thick, contents in few with sandy crystals and prisms of calcium oxalate [Figure 3]c, [Figure 4]a and [Figure 4]b. Collenchymatous hypodermis is 3–5 layered in ridges with cells angularly thickened, while in furrows, it is 3–4 layered and chlorenchymatous. Cortex is scanty, cells polygonal to spherical, large, often with small sandy crystals of calcium oxalate [Figure 3]c, [Figure 4]a and [Figure 4]b. Vascular tissue is made of several vascular bundles arranged in a ring, the smallest 130–238 μm (172) in diameter and largest 184–302 μm (226) in diameter. The vascular bundles are capped by 3–8 layered sclerenchymatous sheath. The vascular ring is enclosed by endodermis of larger polygonal to elongated cells. The vascular bundles are endarch, collateral, and separated by cambium [Figure 3]c, [Figure 4]a and [Figure 4]b. The xylem consists of vessels/tracheids in radial rows, few isolated and interspersed with fibers and few xylem parenchyma cells. The secondary walls of vessels/tracheids are made of scalariform and helical thickenings and a few bordered pitted and annular. The central pith parenchyma consists of large polygonal to spherical cells often with acicular and raphidal needle-like crystals. The pith parenchyma disintegrates and appears hollow at center [Figure 3]c and [Figure 4]a.
|Figure 4: (a) Transection of young stem at the ridge (enlarged) X 146, (b) Transection of stem at furrow region (enlarged) X 432, (c) Transection of mature stem (a portion) X 92. E: Epidermis; ck: Cork; co: Collenchyma; ch: Chorenchyma; p: Parenchyma; c: Cambium; scl: Sclerenchyma; ph: Phloem; x: Xylem; sx: Secondary xylem; pi: Pith|
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Mature stem microscopy
In transection, the structure is almost similar, except the outermost epidermis is replaced by 3–4 layered cork at some places. The cork is often with chloroplasts and is followed by a narrow cortex [Figure 4]c. The secondary xylem is well developed and the phloem is reduced. The vascular cambium is present between xylem and phloem. The interfascicular tissue is replaced by continuous growth of xylem. Pith is hollow [Figure 4]c.
- Pieces of upper epidermis with anomocytic stomata and epidermal cells with straight to curved sides
- Uniseriate macroform conical hair long, either whole or fragments, numerous
- Pieces of lower surface with wavy to sinuate sides and stomata anomocytic and tetracytic types
- Pieces of vessels with scalariform, bordered pits and helical thickenings
- Secretory canals with brownish contents
- Pieces of cortical tissue with attached sclerenchyma.
- Color – Light green
- Taste – Not characteristic
- Odor – Pungent
- Touch – Slightly coarse.
Qualitative phytochemical tests
Loss on drying reveals the presence of water in the plant powder and also some volatile organic matter. Results of physicochemical studies , are summarized in [Table 1] and [Table 2].
High-performance thin layer chromatography fingerprinting
The profile of chromatographic separation scanned at 254 nm reveals six spots [Figure 5] and [Figure 6], out of which one possess maximum composition with Rf at 0.89, while the densitogram scanned at 366 nm revealed nine spots [Figure 7] and [Figure 8], with spot no. 4 and 5 showing maximum composition at Rf0.33 and 0.43, respectively. It is evident from the data that these are characteristic for the studied drug, which will help in identification and authentication of the mother tincture of the drug. These are considered as valuable standards in pharmacopeia. At 254 nm, six spots appear at Rf0.17, 0.33, 0.45, 0.61, 0.76, and 0.89 (all are brown) with various concentrations, while at 366 nm, nine spots appears at Rf0.12 (blue),0.17 (blue), 0.26 (blue), 0.33 (red), 0.43 (blue), 0.56 (red), 0.70 (blue), 0.76 (blue), and 082 (blue). These are again vital fingerprint parameters for the prepared mother tincture and ensure the reliability and reproducibility of the drug.
|Figure 5: High-performance thin layer chromatography densitogram of Erigeron canadensis scanned at 254 nm using chloroform: methanol (9:1 v/v)|
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|Figure 6: High-performance thin layer chromatography video image of Erigeron canadensis at 254 nm in chloroform: methanol (9:1 v/v)|
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|Figure 7: High-performance thin layer chromatography densitogram of Erigeron canadensis scanned at 366 nm using chloroform: methanol (9:1 v/v)|
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|Figure 8: High-performance thin layer chromatography video image of Erigeron canadensis at 366 nm in chloroform: methanol (9:1 v/v)|
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| Discussion|| |
The fresh leaves and stems of E. canadensis L. are used as medicine in Homoeopathy for various therapeutic conditions (Loc. cit). Epidermal cells in the leaf surface show curved to wavy and sinuate sides often with sandy crystals of calcium oxalate as also reported earlier. Stomata occur on either surface with anomocytic, anisocytic, and tetracytic types. Trichomes have been reported as uniseriate and multicellular in the species, which is currently confirmed as uniseriate macroform conical hair. Epidermal cells in transection are found to possess acicular crystals of calcium oxalate in few cells as also reported earlier. Secretory canal is conspicuous and found beneath the vascular bundle of midvein besides close to secondary and tertiary vein bundles of lamina, which confirms the earlier studies.
Stem in transection is rounded with ridges and furrows. Epidermis is single-layered, often containing prismatic crystals of calcium oxalate as reported. The hypodermis is collenchymatous in the ridges and conspicuously possess angular thickening, while furrows have chlorenchymatous cells. The cortical parenchyma possesses some small sandy and acicular crystals and confirms earlier studies. Vascular bundles are several, arranged in a ring covered by a sclerenchymatous cap. The pith parenchyma has large polygonal to spherical cells, often containing acicular or raphidal crystals of calcium oxalate as also earlier reported. The mature stem also shows nearly similar structure, except the epidermis being replaced by 3–4 layered cork at some places. Secondary xylem is well developed; the interfascicular tissue is replaced by secondary xylem. The presence of acicular and raphidal needles of calcium oxalate in the cortex and pith as reported earlier is presently confirmed.
Physical parameters include color, appearance, odor, pH, sedimentation, moisture content, and ash values. Chemical parameters include limit tests, extractive values, and chemical assay; these standards and Rf values are worked out for the first time, form an invaluable data, will serve as a good standardization tool for E. canadensis L.
| Conclusion|| |
The macroscopical, microscopical, and organoleptic characters along with the anatomical and methodology used for the studies are diagnostic and establish the standards.
Dr. Raj K. Manchanda, Director-General, Central Council for Research in Homoeopathy, New Delhi, and Prof. P. Ramachandra Reddy, Project Officer, Drug Standardisation Unit, Hyderabad, for the facilities and encouragements are acknowledged and some help in undertaking HPTLC study by Sreeman Narayana, SRF (C) are acknowledged.
Financial support and sponsorship
Conflict of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2]