Journal of Medicinal and Aromatic Plant Sciences 31 (2009) 42-47
Pharmacognostic and preliminary phytochemical studies on the stem of Terminalia alata Heyne ex Roth. A. SARASWATHY*, K. AMALA, R. SHAKILA, K.N. SUNIL KUMAR Captain Srinivasa Murti Drug Research Institute for Ayurveda and Siddha (CCRAS), Anna Hospital Campus, Arumbakkam, Chennai, India Received : 18th December, 2007
rejuvenative drug used in Ayurveda. It is efficacious in skin diseases, erysipelas, leucoderma, polyuria, cures kapha, haemorrhagic diseases and beneficial for skins and hairs. Bark is used in fever, boils, pruritis, earache, diarrhoea, indolent ulcers and diseases of head. It also has styptic, cardiotonic, astringent, antiseptic bactericidal and demulcent properties. Gum from the trunk is used as a cosmetic and incense [2,9,11,24]. Pharmacognostic and phytochemical studies on stem bark and leaf of T. alata carried out by other workers are available [4,13,16,17]. As there is no detailed pharmacognostical study available for the stem, the present paper attempts to undertake the pharmacognostic, physicochemical, preliminary phytochemical, fluorescence and HPTLC finger print analyses of the stem for the identification of the drug in dried form.
ABSTRACT The present communication attempts to undertake the pharmacognostical and preliminary phytochemical studies on the stem of Terminalia alata Heyne ex Roth., fam. Combretaceae; a large tree with rough bark deeply cracked. It is widely distributed throughout India. Different parts of this plant are used in curing different types of diseases. It is efficacious in skin diseases, erysipelas, leucoderma, polyuria, kapha, haemorrhagic diseases and beneficial for skins and hairs. As there is no pharmacognostic work reported on stem, the macroscopic and microscopic characters are carried out and demonstrated along with their physicochemical parameters, preliminary phytochemical constants and fluorescence analysis. The stem shows cluster crystals of calcium oxalate, thick walled lignified groups of stone cells and pitted medullary ray cells microscopically. The study revealed specific identities for the particular crude drug which will be useful in identification of the raw drug in dried form.
MATERIALS AND METHODS Plant material was collected from Tarikhet, identified with the help of Floras [5,19] and authenticated by NBRI, Lucknow. Dried specimen (No. L/227-St 5) was deposited in the crude drug museum of CSMDRIA, Chennai. Free hand sections were taken, stained with usual methods and the reagents used in pharmacognosy studies [8,23]. Drawings were made with the help of camera lucida.
Key words: Terminalia alata, pharmacognosy, phytochemical studies, Combretaceae.
INTRODUCTION Karumarudu is botanically equated to Terminalia alata Heyne ex Roth. Syn. Terminalia tomentosa W. & A. belonging to the family Combretaceae; a large tree with rough bark deeply cracked. Leaves sub opposite or uppermost alternate, coriaceous, elliptic, oblong, obtuse or even emarginated or slightly acute at apex, flower dull yellow in axillary and terminal panicle spikes, fruit large glabrous or hairy, obovoid – oblong, wings broad striations carried horizontally to the edge [5,19,20]. The tree is widely distributed throughout India, and also reported from Coimbatore, Thirunelveli, Nilgiri, Salem, Tiruchirappalli in South India [24]. It is an important
Powder of the dried stem of T. alata was used for chemical analysis. Physico-chemical studies like total ash, acid insoluble ash, water soluble ash, alcohol and water solubility, loss on drying at 105ºC were carried out as per the WHO guidelines [1,18]. Preliminary phytochemical tests were done as per the standard methods [6,10,12]. The fluorescence behaviour of the powdered drug in the day light and ultra violet light were carried out by moistening the powder in different alkaline and acidic solutions and viewing under the light of different wavelengths in a UV-chamber [3,21]. The HPTLC finger print profile of ethanol extract was performed using precoated silica gel 60 F254 TLC plate (E. Merck) as adsorbent and employing CAMAG Linomat IV automatic sample spotter. The mobile phase used was chloroform: methanol (9:1). The plates after drying were scanned using
* Author for correspondence E.mail:
[email protected];
[email protected]
42
CAMAG TLC scanner (030618) with CATS V 4.06 software at a wavelength of 254 nm using deuterium lamp [7, 14, 15, 22].
CK CT PHF CAM IXPH PI XY PH MR
RESULT AND DISCUSSION Macroscopical characters Stem pieces of the branches, vary in size, cylindrical, nodes swollen, with 4 to 7 protending attached bases of the lateral branches, internodes cylindrical 6 to 11 cm in length, 2 to 4 cm in diameter, finely-longitudinally striated, exhibit at places small lenticels, externally pale brownish, internally pale yellowish, taste somewhat bitter, odour nil. Microscopical characters
Figure 1. Diagrammatic TS of stem
TS of the stem exhibits roughly circular outline, shows on outer about 0.1 mm thick dark brown cork layer in young which often transforms to rhytidoma in older pieces; cortex is parenchymatous about 0.2 mm in thickness with continuous layers of stone cells followed by few rows of brown content cells, pericyclic zone is very narrow about 0.1 mm in thickness traversed with fibres, phloem comparatively wider, about 0.2 to 0.3 mm in thickness traversed with lignified fibres followed by inner xylem occupying the major area of the section which measures about 1.5 mm to 2 mm in thickness. Patches of perimedullary phloem is found encircling the xylem region and there is a parenchymatous pith measuring about 1.2 mm in thickness (Figure 1).
idioblast containing cluster crystals of calcium oxalate, and uni, rarely biseriate medullary rays; the innermost phloem tissue is devoid of fibres, cambium is distinct. Xylem composed of isolated or groups of 2 to 3 vessels encircled with parenchyma which runs at places in tangential bands, fibres are many, medullary rays are pitted, uni to biseriate, in continuation with that of phloem. Pith is lignified, pitted, parenchymatous, encircled with groups of perimedullary phloem (Fig.2-A-C).
SCK ST BCC
Detailed TS shows outer stratified cork formed by alternating bands of small and large rectangular tangentially elongated cells followed by 2-3 rows of narrow, thin-walled, rectangular tangentially running highly lignified continuous patch of stone cells alternating with large radially arranged squarish to rectangular yellowish non-lignified cells followed by few rows of parenchymatous cells filled with brown contents; groups of lignified fibres traversed throughout the pericycle region, in older stem it may not be noticed due to deep development of rhytidoma. Phloem is wider, traversed with tangentially running groups of fibres associated with
CLR
XYV
PF
MR
B
PHF IXPH PH CAM MR PI ______ A
0.1mm
C
______ 0.1mm
Figure-2. Detailed TS of Stem. A. Outer region, B & C. Inner region EXPLANATION FOR THE ABBREVIATION
Plate-1
BCC - Brown content cells; CAM - Cambium; CLR - Cluster Crystals of Calcium Oxalate; CK - Cork; IXPH - Intra Xylary Phloem; MR - Medullary Ray; PF - Pericycle Fibres; PH - Phloem; PHF - Phloem Fibres; PI - Pith; ST - Stone Cell; SCK - Stratified Cork; XY - Xylem; XYV - Xylem Vessel
A. Trunk of T.alata B. Dried stem of T.alata
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Table 1.
Powder analysis Powdered drug is pale yellow in colour with slightly bitter in taste and unpleasant odour. Microscopic examination of the powder shows cluster crystals of calcium oxalate upto 35 ì in diameter, cork in surface view, isolated and groups of lignified thick walled stone cells upto 70 ì in length, lignified pitted parenchyma cells and uniseriate pitted medullary rays; lignified thin-walled fibres with sharp end tips up to 500 ì in length, spiral and pitted vessels upto 350 ì in length, starch grains simple circular to oval in shape up to 25 ì in diameter (Figure 3).
S. No.
Preliminary phytochemical tests for different solvent extracts of stem of T. alata
Test
nChloro- Ethyl Alcohol Hexane form acetate
1
Alkaloid
-
-
-
-
2
Quinone
-
+
+
+
3
Coumarin
-
+
+
+
4
Flavone
+
+
+
+
5
Steroid
+
+
+
+
6
Phenol
-
+
+
+
7
Tannin
-
-
+
+
8
Glycoside/Sugar
-
+
+
+
9
Iridoid
-
-
-
-
10
Terpenoid
+
+
+
+
extract showed the presence of flavone, steroid and terpenoid. Chloroform extract gave positive results for quinone, coumarin, flavone, steroid, phenol, glycosides and terpenoid. Ethyl acetate and alcohol extracts gave positive results for quinone, coumarin, flavone, steroid, phenol, glycosides, terpenoid tannin and gave negative results for alkaloid and iridoid. The results of fluorescence analysis have been presented in Table 2. Quantitative standards revealed that the total ash and water-soluble as content were 2.769 % and 0.619 % respectively. Negligible amount of acid-insoluble siliceous matter (0.9 %) was present in the plant. Amount of tannins and phenol are useful parameters for the identification of the drug. Alcohol and n-hexane (hot) soluble extractive values indicated the presence of polar and non-polar secondary metabolites present in the plant. Water-soluble extractive value was indicating the presence of sugar, acids and inorganic compounds (Table 3). Figure 3. Powder microscopy of Terminalia alata stem; a. cluster crystals of calcium oxalate; b. starch grains; c. spiral vessels; d. brown content cell; e pitted parenchyma; f. cork in surface view; g. groups of stone cells; h. pitted vessels; i. fibres; j. medullary ray
Table 2. Fluorescence analysis of stem of T. alata
S. No.
Powder
1.
Powder
Daylight Pale yellow
Diagnostic characters
UV Yellowish brown
1.
Stratified cork
2.
Powder+1N HCl Pale yellow
Brown
2.
Cluster crystals of calcium oxalate
3.
Powder+50% H2S04
Pale brown
Dark brown
3.
Groups of lignified stone cells.
4.
Dark brown
Uniseriate medullary rays
Powder+1N NaOH
Reddish brown
4.
5.
Powder + Alcoholic 1N NaOH
Pale brown
Yellowish brown
5.
Lignified pitted parenchymatous pith Preliminary phytochemical results showed the presence or absence of certain phytochemicals in the drug. n-Hexane
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Table 3. Physico-chemical parameters of stem of T. alata
S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9.
Parameters
Total ash Water soluble ash Acid-insoluble ash Alkalinity (cc of 0.1N HCl/g) Tannin (% as tannic acid) Phenol (% as gallic acid) Extractive values Water soluble extractive Alcohol soluble extractive n-Hexane-soluble extractive (Soxhlet)
Mean value (%w/w) N=3 + S.D 2.769 + 0.69 0.619 + 0.045 0.069 + 0.059 0.66 + 0.013 6.14 + 0.01 0.52 + 0.004 9.833 + 0.36 7.125 + 0.33 0.548 + 0.002
Figure 5. HPTLC finger print profile of ethanolic extract of Terminalia alata Heyne ex Roth.
TLC (Figure 4) and HPTLC finger print profile of the ethanolic extract, arjunolic and maslinic acids (Figures 5, 6 & 7 respectively) developed in the mobile phase of chloroform : methanol (9:1) and derivatized with vanillin-sulphuric acid reagent showed 9 spots at Rf 0.15, 0.22, 0.28, 0.33 (arjunolic acid), 0.43, 0.53 (maslinic acid), 0.63, 0.83 and 0.91. The identification of spot of arjunolic acid and maslinic acid in the sample solution was confirmed by superimposing the UV absorption spectra (Figures 8,9 respectively) of the test sample with that of standard using CAMAG TLC Scanner.
Figure 6(a). HPTLC finger print profile of maslinic acid
Figure 4. TLC profile of ethanolic extract of Terminalia alata Heyne ex Roth. 1. Sample solution, 2. Arjunolic acid, 3. Maslinic acid
Figure 6(b). HPTLC finger print profile of arjunolic acid
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CONCLUSION Morphology as well as various phamacognostic aspects of the stem sample were studied and described along with phytochemical, physico-chemical, TLC and HPTLC studies in authentification for quality control. Stem of Terminalia alata exhibits a set of diagnostic characters, which will help to identify the drug in dried condition. ACKNOWLEDGEMENT Authors are thankful to the Director of Central Council for Research in Ayurveda and Siddha, New Delhi for financial support. Figure 7. HPTLC finger print profile of maslinic acid.
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Figure 8. UV superimposable spectra of 1. Terminalia alata stem and 2. Arjunolic acid
Figure 9. UV superimposable spectra of 1. Terminalia alata stem and 2. Maslinic acid
46
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