|Year : 2017 | Volume
| Issue : 1 | Page : 3-8
Pharmacognostic evaluation of stem bark and leaves of Anogeissus pendula Edgew
Deeksha Singh1, Manmeet Singh Pannu2, Krishnendra Singh Nama3, Uttam Singh Baghel4, Rakesh Yadav1
1 Department of Pharmacy, Banasthali Vidyapith, Banasthali, Tonk, Rajasthan, India
2 Department of Pharmaceutical Analysis and Chemistry, Khalsa College of Pharmacy, Amritsar, Punjab, India
3 Department of Botany, Career Point University, Kota, Rajasthan, India
4 Department of Pharmacy, Kota College of Pharmacy, Kota, Rajasthan, India
|Date of Web Publication||13-Jul-2018|
Department of Pharmacy, Banasthali Vidyapith, Banasthali, Tonk - 304 022, Rajasthan
Source of Support: None, Conflict of Interest: None
Background: Anogeissus pendula Edgew has ethnomedicinal importance in various parts of India. It is used in diarrhoea, dysentery, cough, wound healing, burns, skin diseases and gastric disorder. No attempts have been made regarding pharmacognostic investigation of the plant till date. Aim: The present study aimed to perform the pharmacognostic study of leaf and stem bark of Anogeissus pendula Edgew. Settings and Design: The study was designed in accordance with standard procedures. Materials and Methods: Pharmacognostic studies viz. organoleptic, macroscopic, microscopic, physicochemical and chromatographic fingerprinting on fresh and dried plant parts along with the hydroalcoholic extracts were conducted. Results and Conclusions: A. pendula has dorsiventral oblanceolate to obovate leaves arranged in opposite pairs while the stem bark has rough appearance and ash to greyish white in colour. Leaves have anomocytic type of stomata. The characteristic microscopic features of leaves were observed to be epidermal cells, palisade and spongy parenchyma, vascular bundles, xylem and phloem cells. Stem bark microscopy revealed the presence of periderm, secondary cortex and secondary phloem. Chromatographic fingerprinting of extracts showed the presence of flavonoids (Rutin and Quercetin) and they were quantified. Rutin was found to be 6.23% w/w in leaves and 9.97% w/w in stem bark extract while Quercetin to be 27.29% w/w and 51.62% respectively. The present study evaluated various pharmacognostic parameters which will help in quality control (standardization) of A. pendula Edgew in crude form, in herbal formulations and also aid in the preparation of an herbal monograph for the species.
Keywords: Anogeissus pendula, fingerprinting, microscopy, organoleptic, physicochemical, standardization
|How to cite this article:|
Singh D, Pannu MS, Nama KS, Baghel US, Yadav R. Pharmacognostic evaluation of stem bark and leaves of Anogeissus pendula Edgew. Ancient Sci Life 2017;37:3-8
| Introduction|| |
Anogeissus pendula Edgew (Family: Combretaceae) popularly known as 'dhok', 'kala dhavda' in Hindi and 'button tree' in English, is a gregarious deciduous shrub or small tree, with pendulous branches but sometimes spiny branches when young. It is native to India commonly occurring in the dry tropical forests and dry mixed deciduous forests of Rajasthan, Madhya Pradesh, Haryana, some parts (Sabarkantha and Banaskantha) of Gujarat and Bundelkhand region (Jhansi, Hamirpur and Banda district) of Uttar Pradesh., It has a maximum height of 9-15 m and 1 m girth. Stem bark appears to be extremely variable, smooth and silvery at first, becoming grey and rough with dark, chapped patches on old trees. Older leaves turn purply-red before being shed between December and February and are renewed in early June with pre-monsoon showers. Flowering occurs in late July to September and of greenish-yellow colour which are massed together in spherical heads with no petals. Fruits form quickly after flowering, ripen and fall between December and early February. They are flat, nearly circular in outline with a long beak, packed together in round heads.,
Various ethnomedicinal uses such as diarrhoea, dysentery, cough, wound healing, skin diseases, burns and gastric disorder of stem bark, seed, fruit and twig have been reported.,,,, Paste of its leaves is applied externally on swellings. It is also reported that the ethyl alcohol extract of aerial parts has diuretic and cardiovascular stimulant potential. The seeds possess haemagglutinating properties against the human A, B and O red cells., Some communities use this multipurpose plant for its fodder, timber and fuel wood. Phytochemical investigations revealed the presence of alkaloid and phenolic compounds such as lignins and tannins., A polyphenolic flavonoid compound isolated, viz. 5, 7, 3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β-D-glucopyranoside, was tested for its neuroprotective effect in transient focal cerebral ischemia in rats and found to be a good neuroprotective agent in stroke as it reduced apoptosis and found to have good antioxidant and anti-inflammatory potential.
Based upon the ethnomedicinal importance of Anogeissus pendula, we have evaluated the pharmacognostic parameters for the first time which will help in the quality control of the herbal formulations containing the same and also to help the preparation of a herbal monograph of the plant in the near future.
| Materials and Methods|| |
Chemicals and instruments
The marker compounds Rutin and Quercetin were procured from Hi-Media laboratories. All the other chemicals used were of analytical grade and were procured from Merck & Co. Inc., Hi-Media laboratories, Sigma-Aldrich and Qualikems Fine Chemical Pvt. ltd. The microscope used to study different microscopic characters was Olympus CX 41.
Fresh leaves and stem bark were collected from Sawai Madhopur (Rajasthan) in the month of November. The plant was identified and authenticated. Collected plant parts were shade dried and powdered coarsely. Standard procedure and analytical grade solvents were used for the extraction. 40 g of plant parts' powder (leaf and stem bark) was preliminary extracted with chloroform and n-hexane, respectively by Soxhlet apparatus and further with the same procedure extracted with hydroalcoholic solution. Obtained extracts were concentrated under reduced pressure and freeze-dried to get yield of 20.1% (leaf – Anogeissus pendula leaf [APL]) and 9. 2% (stem bark – Anogeissus pendula bark [APB]). Extracts were stored in sealed vials in a freezer until tested.
Fresh plant parts were tested for various sensory parameters such as colour, odour, size, shape and taste.
Various macroscopic parameters of fresh leaves such as surface appearance, texture and characters of lamina were recorded. Lamina consists of various distinctive features such as shape, margin, incision, composition, apex, base and venation. Stem bark was studied for various morphological characters such as nature of curvature, surface characteristics and fracture.
For microscopic evaluation, fresh leaves and stem bark were fixed in fixing solution (FA solution containing formalin (5 ml), glacial acetic acid (5 ml) and 70% alcohol (90 ml)) for three days before chloral hydrate solution treatment. Microscopic evaluation was carried out at both levels qualitatively and quantitatively. For qualitative microscopy of leaf and stem bark, a cube of pith was used and fine sections were obtained. Transverse sections of stem bark and leaf as well as longitudinal section of stem bark were studied under microscope. The various characteristic features of unstained sections of stem bark and leaf were studied under microscope and photomicrography was done subsequently. In quantitative microscopy, fresh leaves were used and leaf constants such as stomata number, stomatal index, vein-islet number and vein termination number were studied.
In this study, air dried plant material was used for quantitative determination of physicochemical values such as total ash, acid insoluble ash, water soluble ash, moisture content (loss on drying), foaming index, swelling index, crude fibre, total solid, alcohol extractive value and water extractive value as per standard methods.
Phytochemical investigation of hydroalcoholic extracts of leaves and stem bark was carried out according to standard procedure.
Chromatographic fingerprinting of extracts
High-performance liquid chromatography
HPLC method was developed for Rutin analysis and the following conditions were followed. Analysis was done on LC100 Cyberlab HPLC using Agilent technologies C-18 column with dimensions of 250 × 4.6 mm. HPLC software was Chrom-Workstation HPLC with version number 4-0512-039. Run time for marker compound (Rutin) was 10 minutes and for extracts was 60 minutes. Injection volume was 20μl. Temperature was ambient. Mobile phase and solvents used were sonicated and passed through 0.45μ nylon filter using vacuum pump. Flow rate of mobile phase was kept at 0.8 ml/min. Mobile phase consisted of acetonitrile and 2% acetic acid (v/v) at the ratio of 95:5. Wavelength for analysis was chosen using Shimadzu UV-1800 spectrophotometer and selected as 370 nm.
High-performance thin layer chromatography
HPTLC was performed using a previously used method with some modifications. HPTLC was of CAMAG Linomat 5 “Linomat 5_180745” S/N 180745. Silica gel 60 F 254 HPTLC plates measuring 12 cm × 10 cm manufactured by Merck were used. Software used for HPTLC was WinCATS Planar chromatography manager. CAMAG TLC Scanner “Scanner_180710” S/N 180710 was used for HPTLC analysis. Slit dimensions were 5.00 mm × 0.30 mm. Distance between tracks was 18.0 mm. Scanning speed was 20 mm/s. Dosage speed was 150 nl/s. Predosage volume was 0.2 μl. Band length was set at 10.0 mm. Mobile phase consisted of ethyl acetate: glacial acetic acid: formic acid: water in the ratio of 20:1:1:2.5. Wavelength of HPTLC analysis was set at 254 nm.
| Results|| |
Leaves were small, narrowed at both ends with petiole 1-2 mm long [Figure 1]. They were arranged in opposite pairs. The organoleptic characters of leaves include dark green colour of upper surface and light green colour of lower surface. Leaves had characteristic odour and taste with short appressed hairs above and beneath. Stem bark was rough in appearance and outer surface of ash or grey white colour while the inner surface of faint yellow colour which on exposure to atmosphere turns brown. Stem bark had characteristic odour and with bitter taste.
The morphological characters of leaves were observed as size of 1.5 – 3.1 cm × 0.5 – 1.5 cm. The lamina characters include the shape which was found to be oblanceolate to obovate, entire margin, incision was absent, lamina composition was simple, obtuse to acute apex, cuneate base. The venation was reticulate. Type of arrangement was dorsiventral. On the lower surface there were 5-7 nerves which were ascending and slightly raised. Stem bark had fibrous fracture and variable nature of curvature.
The upper and lower epidermis that is adaxial and abaxial surface respectively, was made up of multiple layers of compactly arranged cells [Figure 2]a. All the epidermal cells were alike and highly cutinized. The stomata were sunken as they were located in a cup shaped depression. Anomocytic type of stomata was found in the leaves [Figure 2]b. Mesophyll was composed of thin walled parenchymatous cells containing numerous chloroplasts. It was differentiated into palisade and spongy parenchyma. The columnar (tubular or cylindrical) palisade cells were arranged compactly with their long axis at right angles to the leaf epidermis. The spongy parenchyma cells were of irregular shape and loosely arranged with large intercellular spaces. These air spaces were connected with the sub-stomatal chamber and maintain gas exchange with the outside through stomata. Adaxial surface was occupied by palisade parenchyma while the abaxial surface was occupied by spongy parenchyma. Veins can be differentiated into a number of size classes and orders. The vein of primary order was thickest and single, occupying median position. Vein further ramified into venules of secondary order that were slightly thinner than the primary vein. The venules further ramified to give rise to veinlets of tertiary order that were the thinnest. The midrib consisted of a typical collateral bundle with an adaxial xylem and abaxial phloem. A thin strip of cambium was also present between xylem and phloem. The vascular tissue of midvein and larger veins had vessels in the xylem and sieve tube in phloem. In smaller veins the xylem was represented by tracheids and the phloem by some sieve elements only. A morphologically distinct group of cells surrounded the vascular bundles called bundle sheath which were thin walled parenchymatous cells extending in the direction parallel to the veins. The parenchymatous bundle sheath extended upto the epidermis on one or both sides of the leaf. It probably plays an important role in the conduction of food material between the vascular bundle and epidermis. Leaf constants viz. stomata number, stomatal index, vein-islet number and vein termination number are presented in [Table 1].
|Figure 2: (a) Transverse section of Anogeissus pendula Edgew leaf (×100); (b) leaf surface showing anomocytic type of stomata (×100). UEp: Upper epidermis, LEp: Lower epidermis, MpC: Mesophyll cell, Xy: Xylem, Pl: Phloem, BS: Bundle sheath|
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Stem bark microscopy
Microscopic characteristics of stem bark are depicted in [Figure 3]. Periderm consisted of cork, phellogen and phelloderm. Cork had 5 – 20 layers which were made up of rectangular, thick walled cells having coloured content. Phellogen had shown 2 – 3 layers and was made up of polygonal and tangentially elongated cells. In phelloderm, cells resembled cells of cortex and were isodiametric with intercellular spaces which were arranged in definite radial rows. Lenticels were also present. Secondary cortex was composed of oval to polygonal cells arranged in 10-15 layers. Secondary phloem was parenchymatous having phloem fibres and sclereids. The cells in secondary phloem were arranged in two systems that were vertical (sieve tubes, companion cells, phloem parenchyma and fibres) and horizontal (ray parenchyma cells formed by the ray initials).
|Figure 3: Transverse section (a) and lateral section (b) of stem bark (×100). C: Cork, SL: Sclerid layer, MR: Medullary ray, F: Fibres, SC: Stone cells, VC: Vascular cambium, Pl: Phloem, CC: Cork cambium, Pd: Periderm|
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Physicochemical analysis of stem bark and leaf powder viz. total ash, acid insoluble ash, water soluble ash, moisture content, foaming index, swelling index, crude fibre, total solid alcohol extractive value and water extractive value are presented in [Table 2].
Preliminary qualitative phytochemical screening revealed the presence of alkaloids, flavonoids, tannins and phenols in both APB and APL.
Chromatographic fingerprinting of extracts
High-performance liquid chromatography
APB gave retention time for Rutin at 4.05 minutes [Figure 4] while APL gave retention time at 4.10 minutes in respect of marker compound Rutin whose retention time was 4.08 minutes. Concentration of Rutin was found to be 9.97% w/w in APB and 6.23% w/w in APL.
|Figure 4: High-performance liquid chromatography chromatograms of (a) Rutin, (b) APB and (c) APL|
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High-performance thin layer chromatography
Quercetin and Rutin concentration was found to be 51.62 and 8.1% w/w in APB whereas 27.29 and 4.40% w/w in APL. Rf for marker compound of Rutin was found to be between 0.06 and 0.13 whereas for marker compound of Quercetin was between 0.74 and 0.88. In case of APB Rf for Rutin was found to be between 0.10 and 0.12 whereas for Quercetin 0.78 and 0.90 while in case of APL it was found to be between 0.04 and 0.10 for Rutin while for Quercetin it was between 0.76 and 0.85. HPTLC chromatograms of Quercetin, Rutin, APB and APL are shown in [Figure 5].
|Figure 5: High-performance thin layer chromatography chromatograms of (a) Rutin, (b) Quercetin, (c) APB and (d) APL|
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| Discussion|| |
Despite its ethnomedicinal importance, no attempt has been made for the pharmacognostic standardization of leaves and stem bark of Anogeissus pendula. The standardization is a vital step in establishing the correct identity, purity, safety and quality of crude drug and it should be established before the inclusion of crude drug in herbal pharmacopoeia. The preliminary material should be under proper control, to maintain the quality of herbal products. Recently, there has been an emphasis in standardization of medicinal plants of therapeutic potential. In spite of modern techniques, pharmacognostic evaluation is still more reliable for identification and evaluation of plants. World Health Organisation (WHO) recommends that the macroscopic and microscopic evaluation is most important in establishing the identity and purity of plants. In the present study, pharmacognostic standardization of leaves and stem bark were carried out for the first time. Morphological, microscopic and physicochemical evaluation provide the simplest, quickest and cheapest means to establish the identity and purity of drugs and also act as reliable tools for detecting adulteration. As the main cause of degradation of desired therapeutic effect of plant species used in traditional systems of medicine is the adulteration of the genuine raw material.,
In physicochemical parameters, ash values and extractive values serve as a reliable aid for detecting adulteration and in identification of plant. Ash values give an idea about inorganic composition or earthy matter or other impurities present along with the drug while extractive values are useful for the determination of exhausted and adulterated drug. Extractive value also helps in indication of chemical constituents and in estimation of specific constituents soluble in particular solvents in a crude drug., Crude fibre content indicates the amount of cellulose, lignin and cork cell. Moisture content below 6% w/w indicates that the crude drug can be stored safely. Foaming index gives an idea about the presence of saponins. Swelling index shows the presence of mucilage. In the present study, the various pharmacognostic parameters such as organoleptic, macroscopic, microscopic, physicochemical and chromatographic fingerprinting have been recorded and those will aid in the quality control and quality assurance of the plant.
Anogeissus pendula has dorsiventral oblanceolate to obovate leaves arranged in opposite pairs while the stem bark has rough appearance and ash to grey white in colour. Microscopy reveals that the leaves have anomocytic type of stomata. The characteristic microscopic features of leaves were observed as epidermal cells, palisade and spongy parenchyma, vascular bundles, xylem and phloem cells. The columnar (tubular or cylindrical) palisade cells were arranged compactly with their long axis at right angles to the leaf epidermis. This arrangement of palisade cells protects chloroplasts from excessive heat of sun rays. Stem bark microscopy revealed the presence of periderm (cork, phellogen and phelloderm), lenticels, secondary cortex, secondary phloem (sieve tubes, companion cells, phloem parenchyma, fibres, ray parenchyma cells).
Physicochemical evaluation shows that the total ash is higher in stem bark than the leaves and same results replicate in case of water soluble ash but the acid insoluble ash is higher in leaves. Moisture content is below 6% w/w for both stem bark and leaves and therefore they can be stored safely. Foaming index reveals very low quantity of saponins but higher in bark than leaves. Both the extracts of plant parts showed higher water extractive value than alcohol extractive value.
Chromatographic fingerprinting supports the findings of preliminary phytochemical evaluation and reveals the presence of flavonoids such as Rutin and Quercetin in both the extracts but APB that is hydroalcoholic extract of stem bark, has more quantity of active phytochemicals than the APL.
| Conclusion|| |
In conclusion, the parameters which are evaluated here can be considered as unique enough to identify and decide the authenticity of A. pendula which will serve in the development of pharmacopoeial standards for future studies.
Authors are thankful to Dr. Krishnendra Singh Nama and Dr. Kiran Chaudhary, Botanist, Maa Bharti P. G. College, Kota for the identification, collection and authentication of the plant.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]