| Abstract|| |
Careya arborea Roxb (Lecythidaceae) is known as Kumbhi in Ayurveda and is an important medicinal plant. It has a large panel of indication and would be more suitable to evaluate the bark since it has been used in Ayurveda in treatment of tumours, cough, bronchitis, haemorrhoids, intestinal worms, dysentery, ulcers and eruptive fevers, particularly small pox to name a few. The present study provided taxonomic characters of the plant, pharmacognostical and physicochemical details of the stem bark which will help in laying down pharmacopoeial parameters. Many important diagnostic characters such as lenticular openings at certain places of bark; presence of tanniferous cells in cork, secondary cortex and secondary phloem; layers of stone cells in secondary cortex and secondary phloem; presence of fibroid sclereids; rhomboidal, prismatic type of crystals in secondary phloem and presence of starch grains in medullary rays will certainly help in the identification of the drug. The preliminary phytochemical analysis revealed the presence of carbohydrates and glycosides, phenolic compound and tannins in ethanol and aqueous extracts, saponin glycosides in aqueous extract and phytosterols are present in petroleum ether, benzene, acetone and ethanol extracts. Fixed oil and fats are found in petroleum ether and benzene. Flavonoids, gums and mucilage, proteins and amino acids were totally absent in all extracts. HPTLC profile established for the plant will help in identification of the drug and also in isolating and identifying the biomarker compound responsible for the bioactivity.
Keywords: Careya arborea , HPTLC, Kumbhi, Pharmacognostical, physicochemical
|How to cite this article:|
Goyal KK, Satish Kumar B N, Mruthunjaya K, M, Yoganarasimhan S N. Pharmacognostical studies of stem bark of Careya arborea Roxb. Int J Green Pharm 2011;5:6-11
|How to cite this URL:|
Goyal KK, Satish Kumar B N, Mruthunjaya K, M, Yoganarasimhan S N. Pharmacognostical studies of stem bark of Careya arborea Roxb. Int J Green Pharm [serial online] 2011 [cited 2013 Jun 19];5:6-11. Available from: http://www.greenpharmacy.info/text.asp?2011/5/1/6/82088
| Introduction|| |
Careya arborea Roxb. which is popularly known as Kumbhi in Ayurveda is distributed throughout India, Sri Lanka and Malaya peninsula.  The accepted botanical source of Kumbhi is C. arborea Roxb.  The plant is reported as fish poison,  antileishmanial,  antitumour, antioxidant  and antidiarrhoeal.  In spite of abundant uses, the pharmacopoeial standards of C. arborea stem bark is not been explored. In the present study, the pharmacognostic parameters of the stem bark of C. arborea is studied, such as exomorphological, histomorphological, powder microscopy, quantitative microscopy and physical standards like ash values, extractive values, loss on drying, preliminary organic analysis and chromatographic studies.
| Materials and Methods|| |
Fresh stem barks were collected from the vicinity of Tirunelveli, Tamil Nadu, during December, 2007, preserved in 2% formalin for histological studies. Botanical identification was carried out using local floras , and authenticated by Dr. S. N. Yoganarasimhan, Plant Taxonomist and Visiting Professor M.S Ramaiah College of Pharmacy, Bangalore. Voucher herbarium specimen  (028) is preserved along with crude drug sample at the herbarium of M. S. Ramaiah College of Pharmacy, Bangalore.
Pharmacognostical evaluation including histochemical study was carried out by taking free-hand sections according to Johansen  and Wallis  and powder studies according to Evans.  The Kannada vernacular name is provided following Gurudeva. 
Photomicrographs were obtained by observing free-hand sections of drug under compound binocular microscope (Olympus-CH20i) with built-in analogue camera (CMOF, 1.4 mega pixel). Computer images were captured using AV Digitaliser having Grand VCD 2000 Capture Guard. Measurements of cells and tissues were carried out using Micro Image Lite Image Analysis Software (Cybernetics, Maryland, USA). Physicochemical constants, organic analysis, ultra-violet analysis and chromatographic studies were carried out from shade-dried powder following prescribed methods. ,,,,
HPTLC studies were carried out on ethanol extract using Camag HPTLC system equipped with Linomat V sample applicator, Camag TLC scanner 3 and CATS 4 software for interpretation of data. An aluminium plate (5×10 cm) precoated with Silica Gel 60F254 (E Merck, name of the place) was used as adsorbent. The plates were developed for alcohol extract by using mobile-phase ethyl acetate : methanol : water (30 : 05 : 04) and for aqueous extract by using mobile-phase n-hexane : ethyl acetate (9 : 1). Post-derivatization was done by dipping in anisaldehyde H 2 SO 4 reagent. Scanning was done under 254, 366 and 425 nm.
| Results|| |
C. arborea Roxb.; Pl. cor. t. 218. 1811; Gamble, fl. Pres. Madras 1:345. 1957 (repr.ed). Vernacular name - Doddaale (Kannada). A glabrous middle-sized or large tree attaining 20 m, barks are thick, rough, dark-grey. Leaves are crenate-denticulate, obovate, shortly acuminate, 10 to 30×5 to 15 cm, glabrous, tapering at base, main nerves 10 to 12 pairs; petioles 0 to1.8 cm long, stout, margined. Flowers are 6 to 9 cm across, yellowish-white, foetid, sessile, thick, swollen, hard, terminal spikes. Fruits are 6.3 to 7.5 cm in diameter, globose, green, glabrous, crowned with calyx-segments and style scars. It is distributed in deciduous forests and grasslands up to an altitude of 1500 m and also found in Andaman Islands and widely found in India, Sri Lanka, Malaya Peninsula, occasionally planted in gardens and along roadsides.
The stem bark is brownish colour; rectangular shape, curved; Surface: outer surface with lenticular openings at certain places, rough; inner surface is reddish brown, smooth; Size: pieces of 2.5 to 7 cm long; Fracture: fibrous, not peeling off easily; Taste: acrid; Odour: without characteristic.
Transverse section of the bark showed a cork region followed by a wide secondary cortex and a secondary phloem region. A few layers of rhytidome were found as the outermost region of the cork. Lenticular openings were found at intervals. Cork was multilayered, consisting of rectangular parenchymatous cells, measured (11.93-14.30-19.04)×(4.03-4.59-6.06) μm; a few outer layers contained tanniferous cell content. Next to the cork was found a wide multilayered secondary cortex. The cells of secondary cortex were collenchymatous, measured (13.63-20.24-27.37)×(6.23-10.49-14.67) μm. Some cells are found to contain tannins, cell measured (8.82-13.89-17.43)×(8.62-9.30-10.01) μm and some other oil content, stone cells were found distributed either singly or in groups, sometimes running tangentially, in 3 to 4 layers. Vascular traces containing vascular bundles were found distributed here and there in the secondary cortex. Next to the secondary cortex was found a broad secondary phloem region. Groups of stone cells in tangential rows were found distributed in secondary phloem. Phloem consists of phloem parenchyma, companion cells and sieve tubes. Medullary rays were biseriate or uniseriate. Rays cells contained tannin, some cells were found to contain simple starch grains of size (1.93-3.23-4.67) μm either solitary or in groups; some cells of secondary phloem contain rectangular, rhomboidal or prismatic type of crystals that measured (10.50-14.30-17.00) μm [Figure 1].
Macerated section exhibited following characters [Figure 2]:
- Cork cells which are thick walled, parenchymatous, some were reddish brown and measured (13.40-18.37-24.90)×(10.57-13.59-17.69) μm.
- Secondary cortex cells containing yellowish brown content. Phloem parenchyma cells with thin walled measured (28.20-31.66-36.07)×(20.21-20.78-21.12) μm.
- Fibres of different size and shape with broad lumen and narrow pointed ends; they were septate or bundled, simple or forked, measured (78.94-228.63- 528.82)×(6.37-13.47-20.23) μm.
- Stone cells in groups with broad lumen and concentric striations measured (27.36-29.46-32.39)×(15.64-19.77- 22.46) μm.
- Fibroid sclereids with unequal arms and of different size and shape.
- Starch grains which are simple, oval or ellipsoid, turning blue when treated with iodine, measured (17.34-17.69-18.04) μm.
- Crystals of calcium oxalate, rhomboidal in shape, measured (10.74-18.53-27.81) μm.
The powder of C. arborea was reddish yellow, without characteristic odour and with acrid taste.
When powder was mounted with chloral hydrate, phloroglucinol and HCl or with safranin the following elements were observed:
- Parenchyma cells which are rectangular and with cell content.
- Fragments of fibres which are thick walled.
- Fibroid sclereids of different sizes and shapes.
- Crystals of calcium oxalate rhomboidal and prismatic in shape.
- Starch grains which are rounded and simple, turning blue when treated with iodine.
The sections of stem bark of C. arborea Roxb when treated with phloroglucinol and dil. HCl gave red colour indicating the presence of lignin; with ferric chloride turned bluish black showing presence of tannins; with con. HCl effervescence was observed showing the presence of crystals; when treated with iodine gave bright blue colour showed presence of cellulose; with ruthenium red produces red colour and with Dragendorff's reagent, no brown colour was observed indicating presence the of starch, mucilage and absence of alkaloids, respectively.
Physicochemical Parameter of Powder of Stem Bark of C. arborea
Physicochemical parameter value (% w/w)
The percentage moisture content was found to be 11.5% w/w; total ash, acid insoluble ash and water-soluble ash determined were 4.5067, 0.170 and 1.10% w/w, respectively. Water-soluble extractive (hot) value was 15.8; alcohol-soluble extractive (cold) value was 15.99 and 10.77% w/w foreign organic matter was 1.2612% w/w; the colour, consistency and % yield of successive extractive values of powder were petroleum ether (60-80 o C) (pale brown, sticky mass, 0.92), benzene (pale brown, sticky mass, 1.66), chloroform (brownish black, sticky mass, 0.58), acetone (reddish brown, semisolid mass, 3.42), ethanol (reddish brown, semi solid, 2.06), water (reddish cream, semi solid, 0.64).
Preliminary organic analysis
A known quantity of dried powder was extracted in a Soxhlet with petroleum ether (60-80 o C), benzene, chloroform, acetone and ethanol and finally macerated with chloroform-water (2%) for 24 hours successively and tested for different constituents; it revealed the presence of carbohydrates and glycosides in ethanol and aqueous extract, saponin glycosides were found in aqueous extract, phenolic compound and tannins were found in ethanol and aqueous extract, phytosterols were present in petroleum ether, benzene, acetone and ethanol extracts. Fixed oil and fats were found in petroleum ether and benzene. Alkaloids, flavonoids, gums and mucilage, proteins and amino acids were totally absent in all extracts.
HPTLC profile for both alcohol and aqueous extract of the stem bark was carried out. At 254 nm, aqueous extracts showed 7 phytoconstituents at Rf 0.15, 0.26, 0.41, 0.45, 0.48, 0.54, 0.94, out of which spots at the Rf 0.15 is pronounced and all other spots were least pronounced and peaks at 0.15, 0.54 are least pronounced. Under 366 nm, aqueous extract revealed 5 phytoconstituents at Rf 0.22, 0.42, 0.67, 0.70, 0.96, out of which spots 0.22 is pronounced and peaks at 0.42, 0.67, 0.70 were least pronounced. Under 425 nm, aqueous extract revealed 2 phytoconstituents at Rf 0.17, 0.98, out of which all spots were least prominent.
After derivatization, the aqueous extract revealed 16 phytoconstituents at Rf 0.13, 0.26, 0.29, 0.34, 0.36, 0.38, 0.42, 0.49, 0.52, 0.55, 0.59, 0.63, 0.66, 0.81, 0.86, 0.94. Out of these, no spots were pronounced under 254 nm. After derivatization, the aqueous extract revealed 14 phytoconstituents at Rf 0.11, 0.15, 0.22, 0.26, 0.33, 0.38, 0.43, 0.53, 0.55, 0.59, 0.72, 0.81, 0.86, 0.89, out of which spots corresponding to Rf 0.15, 0.26, 0.43 were pronounced and other spots were least pronounced under 366 nm. After derivatization, the aqueous extract revealed 10 phytoconstituents at Rf 0.11, 0.15, 0.26, 0.33, 0.64, 0.77, 0.81, 0.85, 0.86, 0.97, out of which 0.86 spot was prominent and all other peaks were not prominent under 425 nm.
Under 254 nm, alcohol extract revealed 4 phytoconstituents at Rf 0.12, 0.75, 0.83, 0.91, out of which spots at the Rf 0.12 was prominent, whereas spots at the Rf 0.75, 0.83, 0.91 were less prominent and peaks at Rf 0.83, 0.91 were least pronounced. Under 366 nm, ethanol extract revealed 9 phytoconstituents at Rf 0.10, 0.21, 0.31, 0.36, 0.44, 0.49, 0.68, 0.79, 0.90, out of which spots Rf 0.21, 0.68, 0.79, 0.90 were pronounced and peaks at 0.68, 0.79 were least pronounced. Under 425 nm, alcohol extract revealed 4 phytoconstituents at Rf 0.12, 0.73, 0.75, 0.82, out of which no spots were prominent and peaks at 0.73, 0.75, 0.82 were least pronounced.
After derivatization, the alcoholic extract revealed 24 phytoconstituents at Rf 0.13, 0.18, 0.21, 0.26, 0.29, 0.31, 0.37, 0.42, 0.47, 0.50, 0.53, 0.55, 0.57, 0.61, 0.65, 0.69, 0.71, 0.74, 0.77, 0.80, 0.81, 0.85, 0.87, 0.91, out of which spots at the Rf 0.46 was most prominent spots and all other spots were found least prominent under 254 nm. After derivatization, the alcoholic extract revealed 12 phytoconstituents at Rf 0.11, 0.20, 0.23, 0.25, 0.31, 0.38, 0.47, 0.50, 0.58, 0.62, 0.71, 0.92, out of which spots at the Rf 0.11, 0.38, 0.62, 0.71, 0.92 were found more prominent under 366 nm. After derivatization, the alcoholic extract revealed 15 phytoconstituents at Rf 0.13, 0.18, 0.21, 0.24, 0.29, 0.40, 0.45, 0.50, 0.56, 0.74, 0.77, 0.81, 0.87, 0.95, 0.97, out of which spots at the Rf 0.87 was more prominent than the other peaks under 425 nm [Figure 3] and [Figure 4].
Ultraviolet analysis of powdered drug under ultraviolet and ordinary light when treated with different reagents emitted various colour radiations which help in identifying the drug in powder form [Table 1].
| Discussion|| |
The evaluation of a crude drug is an integral part of establishing its correct identity. Before any crude drug can be included in herbal pharmacopoeia, pharmacognostical parameters and standards must be established. Therefore, some diagnostic features have been evolved to identify and to differentiate the C. arborea stem bark and from the other crude drugs and adulterants. In this regard, the important microscopic features of the stem bark were documented; Macerate studies and powder analysis exhibits the elements like presence of tanniferous cells in the cork, secondary cortex and secondary phloem, layers of stone cells in secondary cortex and secondary phloem, presence of fibroid sclereid, presence of rhomboidal, prismatic type of crystals in secondary phloem, presence of starch grains in medullary rays. Histochemical test shows the presence of lignin, cellulose, tannins, starch, mucilage and crystals. Physicochemical parameter values like moisture content, total ash, acid insoluble, water-soluble ash, extractive values, foreign organic matter, the colour, consistency and percentage of successive extractive values were determined. The preliminary phytochemical analysis revealed the presence of carbohydrates and glycosides, phenolic compounds and tannins in ethanol and aqueous extract, saponin glycosides in aqueous extract, phytosterols in petroleum ether, benzene, acetone and ethanol extracts. Fixed oil and fats are found in petroleum ether and benzene. HPTLC profile of ethanol and aqueous extracts showed a number of phytoconstituents present in them. Thus, pharmacognostical studies and phytochemical screening can serve as a basis for proper identification, collection and investigation of the plant. These parameters, which are being reported, could be useful in the preparation of the herbal monograph for its proper identification and evaluation.
| Conclusion|| |
As there is no pharmacognostical anatomical work on records for this traditionally much valued herb, present work is taken up in the view to lay down the macroscopic and microscopic standards, which could be used in deciding the genuineness of the herb, irrespective of their collection from different sources. The coloured photographs of the stem bark of the above-mentioned plants might facilitate the researcher for identification. The results of the phytochemical screening, chemomicroscopical tests and fluorescence behaviours of the powder of the stem bark can be considered as distinguishing parameters to identify and decide the authenticity of C. arborea and thus can be used as standards for reference purpose also. The outcome of the quantitative parameters described on the above-mentioned plant parts (stem bark) might be useful in determining the authenticity of the drugs. HPTLC profile helps in standardisation and also for undertaking work on isolating and identifying the bioactive compound(s).
| Acknowledgement|| |
The authors are thankful to the Gokula Education Foundation and to the Principal, M. S Ramaiah College of Pharmacy and V. V. Pura College of Science, Bangalore for evincing interest in this work. They are also thankful to Sri. V. Chelladurai of Tirunelveli for help in providing authentic plant material.
| References|| |
|1.||Yoganarasimhan SN. Medicinal Plants of India: Vol 1. Karnataka, Bangalore; Interline Publishers; 1996. p. 95-6. |
|2.||Yoganarasimhan SN. Medicinal plants of India vol.2: Tamil Nadu. Bangalore; Cybermedia; 2000. p. 263. |
|3.||Kumar V, Sikarwar RL. Plants used as fish poison by tribals of Surguia district in Chhattisgarh, India. Ethnobotany 2003;15:87-9. |
|4.||Mandal D, Panda N, Kumar S, Banerjee S, Mandal NB, Sahu NP. A triterpenoid saponin possessing antileishmanial activity from the leaves of Careya arborea. Phytochemistry 2006;67:183-90. |
|5.||Natesan S, Badami S, Dongre SH, Godavarthi A. Antitumor activity and antioxidant status of the methanol extract of Careya arborea bark against Dalton's lymphoma ascites-induced ascetic and solid tumor in mice. J Pharmacol Sci 2007;103:12-23. |
|6.||Rahman MT, Khan OF, Saha S, Alimuzzaman M. Antidiarrhoeal activity of the bark extract of Careya arborea Roxb. Fitoterapia 2003;74:116-8. |
|7.||Fischer CEC: Gamble JS. The flora of the presidency of Madras. Vol 1. Dehradun: BSI (reprinted); 2005. p. 102-13. |
|8.||Henry AN, Chithra V, Balakrishnan NP. Flora of Tamil Nadu, Series 1. Coimbatore: Botanical Survey of India; 1989. p. 54. |
|9.||Jain SK, Rao RR. Field and herbarium methods. New Delhi: Today and Tomorrow's Printers and Publishers; 1977. p. 22-61. |
|10.||Johansen DA. Plant Microtechnique: New York: McGraw Hill; 1940. p. 183-203. |
|11.||Wallis TE. Text Book of Pharmacognosy, Delhi: CBS Publisher and Distributor; 1985. p. 352-7 |
|12.||Evans WC. Trease and Evans Pharmacognosy, 15th ed. London: Saunders; 2002. p. 523-5. |
|13.||Gurudeva MR. Botanical and Vernacular Names of South Indian Plants. Bangalore: Divyachandra Prakashana; 2001. p. 305. |
|14.||Kokoski JC, Kokoski RJ, Slama FJ. Fluorescence of powdered vegetable drugs under ultraviolet radiation, J Am Assoc 1958;47:715. |
|15.||Anonymous. Indian Pharmacopoeia. New Delhi: Controller of Publications; 1996. p. A-7,A-97. |
|16.||Wagner H, Bladt S. Plant Drug Analysis. Berlin: Springer; 1996. p. 64, 83. |
|17.||Harborne JB. Phytochemical Methods. 3rd ed. London: Chapman and Hall; 1998. p. 85-8. |
|18.||Sethi PD. High Performance Thin Layer Chromatography. 1st ed. New Delhi: CBS Publishers and Distributors; 1996. p. 3-4. |
Kamal Kumar Goyal
Department of Pharmacognosy, Sri Balaji College of Pharmacy, Jaipur, Rajasthan
[Figure 1], [Figure 2], [Figure 3], [Figure 4]