Indian Journal of Natural Products and Resources Vol. 2(2), June 2011, pp. 211-217
Pharmacognostical studies and preliminary phytochemical investigations on the bark of Persea macrantha (Nees) Kosterm (Lauraceae) Y A Kulkarni1*, S B Gokhale1, S U Yele1, S J Surana2 and A U Tatiya2 1
School of Pharmacy & Technology Management, SVKM’s NMIMS, Mumbai-400 056, Maharashtra, India Department of Pharmacognosy, R.C.Patel Institute of Pharmaceutical Education and Research, Shirpur-425 405, Dist.-Dhule, Maharashtra
2
Received 30 June 2010; Accepted 25 November 2010 Persea macrantha (Nees) Kosterm (Lauraceae) is an important traditional medicinal plant being used for treatment of asthma and rheumatism. Systematic pharmacognostical evaluation of bark of the plant has been carried out with respect to macroscopy, microscopy, physicochemical parameters and estimation of different standards. TLC profiles were developed for petroleum ether and chloroform extract. The preliminary phytochemical investigations indicated presence of alkaloids, tannins, carbohydrates and steroids. The results obtained from standardization of bark established the macro- and microscopical parameters, physicochemical parameters, TLC profiles that characterize the genuine plant drug (P. macrantha). These parameters can be utilized for quick identification of the drug and are particularly useful in the case of powdered materials. Keywords: Lauraceae, Machilus macrantha, Persea macrantha, Pharmacognosy, Phytochemical. IPC code; Int. cl. (2011.01) A61K 36/54, A61K 129/00
Introduction Persea macrantha (Nees) Kosterm syn. Machilus macrantha Nees (Family Lauraceae) is up to 27 m tall tree and 3 m in girth with cylindrical bole up to 7.5 m long (Plate 1). Leaves are oblong to ellipticlanceolate, flowers are yellow in panicles in upper axils and fruits are ovoid, smooth, dark green1. It is found in Western Peninsula, Sri Lanka and in India up to an altitude of 2100 m. In India it is locally known as Golum, Pisara, Kurma, Gulmavu and Chittutandrimara in various regions. The tree has many folk uses in various states of India; the bark is used in consumption, asthma and rheumatism2 while the leaves are used externally in ulcer3. Gaind & Baveja has reported detail pharmacognostical studies of root4. The alcoholic extract of the limed roots indicated presence of alkaloids, machiline and macranthine are the major alkaloids isolated from the roots5,6. The isolated alkaloid, machiline showed marked hypotensive effect in test animals7. Some lignans like machicendiol, machicenonol and machicenin have been isolated from leaves of the plant8,9. —————— *Correspondent author: E-mail:
[email protected]; Phone: +91 9930030548
Crude extracts of the bark have been studied for anti-inflammatory activity10 and antiarthritic activity11. Detail pharmacognostical studies of leaf of the plant have been reported by Kulkarni et al12. Since there is no report of systematic pharmacognostical and phytochemical studies on bark, in order to fix some standards for its
Plate 1 Persea macrantha tree
212
INDIAN J NAT PROD RESOUR, JUNE 2011
identification, this study was planned to study detailed pharmacognosy and preliminary phytochemistry of the same.
Powder characteristics
Preliminary examination, behaviour of powder with different chemical reagents and microscopical examination was carried out16.
Materials and Methods Physicochemical parameters
Plant material
The fresh bark of P. macrantha was collected in the month of April from Lonavala, Dist. Pune (MS), India. It was identified and authenticated by the scientists of Botanical Survey of India, Pune, India and a voucher specimen (b12) of the bark (Plate 2) is deposited in department for future reference. Macro- and Microscopy
Macroscopic examination of the bark was carried out according to standard procedures13,14. Fresh bark was selected for the microscopical studies. Sections were cut on a microtome and by free hand sectioning. Numerous temporary and permanent mounts of the microscopical sections of the bark specimen were made and examined microscopically. Histochemical tests were carried out using hydrochloric acidphloroglucinol to reveal lignified elements, iodineiodide for starch, Sudan IV for lipophilic substances, Dragendorff’s reagent for alkaloidal substances, ruthenium red for mucilage and ferric chloride for phenolic compounds15,16. Photomicrographs of the microscopical sections were captured with the help of Motic photomicroscope (Canada) provided with Motic Images Plus 2.0 software.
Percentage of total ash, acid-insoluble ash, water-soluble ash, sulphated ash and loss on drying were calculated as per the Indian Pharmacopoeia17. The total ash of the powdered bark was tested for different inorganic constituents18. Various extracts were prepared for the study of extractive values of the bark19. Fluorescence analysis of powdered bark was carried out by standard methods20-22. Preliminary phytochemical analysis
For the preliminary phytochemical analysis, 100 g of powdered bark of P. macrantha was extracted with petroleum ether (40-60°C), chloroform, ethyl acetate and methanol successively, using Soxhlet apparatus. The aqueous extract was prepared by cold maceration technique. The extracts were concentrated under vacuum using rotary vacuum evaporator, dried and weighed. Each extract was tested for presence of different phytoconstituents, viz. triterpenoids, steroids, alkaloids, sugars, tannins, glycosides, flavonoids, proteins and amino acids by usual prescribed methods23,24. The TLC pattern of petroleum ether extract and chloroform extract was studied using pre-coated silica gel plates (Merck).
Plate 2 Bark of Persea macrantha
KULKARNI et al: PHARMACOGNOSY OF PERSEA MACRANTHA BARK
Quantification of total phenolics
The concentration of phenolic compounds in powdered bark was determined by Folin-Ciocalteu colorimetric method25. The absorbance was measured at 765 nm against a reagent blank, using Shimadzu UV-1601 spectrophotometer. The total polyphenolic content was calculated as gallic acid equivalents and expressed in % as gallic acid. Quantification of total tannins and carbohydrates
Total tannins were estimated by using Folin-Denis method26. The absorbance was measured at 760 nm using Shimadzu UV-1601 spectrophotometer. Analyses were carried out in triplicate and the quantification was calculated from a calibration curve obtained with tannic acid. Total tannins were expressed as g/100 g of tannic acid equivalent. Total carbohydrates were estimated by using phenol-sulfuric acid method27. The absorbance of the solution was measured at 490 nm using Shimadzu UV-1601 spectrophotometer. Glucose was used as a standard to obtain a calibration curve. Estimation of total alkaloids, mucilage, unsaponifiable matter and crude fibre content
The total alkaloids and mucilage content was determined by the prescribed method28 and the unsaponifiable matter of the bark was determined by the standard procedure of Indian Pharmacopoeia17. The total crude fibre content of the bark was determined by the procedure described by Raghuramulu et al29. Results and Discussion Macroscopy
The bark is externally brownish and internally light reddish brown in colour. It occurs in curved or sometimes flat pieces with size of 7-8×18-20 cm and thickness of about 1.5-3 cm (Plate 2). It has mucilaginous taste which is followed by bitter sensation. The odour is characteristic, unpleasant and has slight astringent effect on throat. The bark shows fibrous fracture. The bark shows number of masses of moss and fungal growth. Outer surface of bark has got numerous lenticels. Number of wrinkles and undulations are also seen on outer surface, while inner surface shows presence of numerous striations. The bark is smooth and has glistering appearance due to numerous shining calcium oxalate crystals in sunlight. Overall the bark is compact, hard and lighter in weight. Fresh
213
bark detached from trunk of the tree is yellowish and turns to brown and then reddish brown on storage. Microscopical characteristics
Transverse section of bark showed roughly four regions- periderm, cortex, a band of sclerenchyma and secondary phloem (Plate 3a). Periderm is composed of cork, phellogen and phelloderm (Plate 3b). Cork is stratified and consists of several layers of radially arranged rows of thin walled, elongated cells. Phellogen is made up of one to two layers of thin walled, rectangular, lignified cells. Phelloderm is one to two layered in thickness and exhibits thin walled, non-lignified, brown coloured and roughly rectangular cells. Secondary cortex is composed of ten to thirteen layers of parenchymatous cells (Plate 3c), encircling either isolated or groups of scattered sclereids. Each sclereid is more or less rectangular in shape and pitted with thickened inner and radial walls. Some of cortical cells contain prismatic, microsphenoidal calcium oxalate crystals and simple starch grains. A continuous, well-developed layer of sclereids (sclerenchymatous band) separates the cortex and secondary phloem region. The inner and radial walls of the sclereids are thicker than the outer walls giving the appearance of the letter ‘U’. Groups of small pericyclic fibres are found on the outer side of sclerenchymatous band (Plate 3d). Secondary phloem region contains phloem parenchyma, phloem fibres and medullary rays (Plate 3e). Phloem parenchyma consists of thin walled cells containing starch and calcium oxalate crystals. Numerous mucilage cells were seen in phloem parenchyma. Phloem fibres occur in group of 2-3, embedded in phloem parenchyma. These are mostly circular and lignified. Medullary rays which are generally 1-3 cells wide divide radially the phloem parenchyma. The results of histochemical tests are given in Table 1. Powder characteristics Macroscopic
The powder is brown in colour, smooth in texture, bitter and with characteristic odour. After addition of small quantity of water, a mucilaginous mass was formed which indicates presence of considerable amount of mucilage. After pressing a little amount of powder between filter paper, no greasy stain
214
INDIAN J NAT PROD RESOUR, JUNE 2011
Plate 3 Microscopical structure of Persea macrantha bark: (a) Transverse section, (b) periderm, (c) cortex, (d) sclerenchyma, (e) secondary phloem. Abbreviations— pd-periderm, co-cortex, scl-sclerenchyma, ph-phloem, ck-cork, phl-phellogen, phd-phelloderm, mr-medullary rays, pp-phloem parenchyma pf-pericyclic fibers.
KULKARNI et al: PHARMACOGNOSY OF PERSEA MACRANTHA BARK
215
Table 1 Histochemical tests on transverse sections of Persea macrantha bark Reagent
Constituent
Phloroglucinol+ hydrochloric acid Lignin Aniline sulphate + sulphuric acid Lignin Conc. Sulphuric acid Cellulose Sudan III solution Oil globules Aqueous Ferric chloride Tannins Dragendorff’s reagent Alkaloids Libermann-Burchardt reagent Steroids SbCl3 Steroids Millons reagent Ruthenium red Mucilage Weak Iodine solution Starch Caustic alkali + HCl Ca. oxalate Note: +++ High, ++ Moderate, + Slight, - Negative
Colour
Tissue
Degree of intensity
Pink Yellow Green Black Light orange Greenish Reddish Pink Blue Green
Periderm, Stone cell layer, Cortex, phloem Periderm, Stone cell layer, Cortex, Phloem Cortex Cortex Cortex Cortex Cortex Phloem Cortex, phloem Cortex, phloem
+++ +++ +++ +++ +++ ++ + ++ ++ ++
Table 2 Behaviour of bark powder with different chemical reagents Reagent
Colour/ Precipitate
Constituent
Conc. sulphuric acid Aqueous ferric chloride (5%) Iodine solution Picric acid solution Aqueous mercuric chloride solution Magnesium- hydrochloric acid Aqueous silver nitrate solution Ammoniacal solution Aqueous potassium hydroxide (5%)
Reddish Blackish Blue Yellowish Brownish No change No ppt. No change No change
Steroids present Tannins present Starch present Alkaloids present Alkaloids present Flavonoids absent Proteins absent Anthraquinone glycosides absent Anthraquinone glycosides absent
was found, indicating absence of fatty oils. After shaking the powder with water in a test tube, no persistentfoam was formed indicating absence of saponins. Behaviour of powder with different chemical reagents is shown in Table 2. Microscopic
The powder showed presence of fibres which are lignified, elongated, tubular, narrow, pointed and isolated phloem fibres with simple or branched pores (Plate 4). The stone cells are found in groups or isolated. They are roughly rectangular or oval in shape, lignified with thick walls and pitted in nature. The cork cells are polygonal, appear brownish in colour. The powder showed abundant, microsphenoidal and prismatic calcium oxalate crystals (35-40 µ). The starch grains are simple, adequate having size of about 26-29µ.
Table 3 Percentage of ash, loss on drying and extractive values of Persea macrantha bark Parameter
% w/w (Mean a± SEM)
Total ash Acid insoluble ash Water soluble ash Sulphated ash Loss on drying Petroleum ether extractive Chloroform extractive Ethyl acetate extractive Ethanol extractive Water extractive a Mean value of three readings.
5.53 ± 0.27 0.54 ± 0.02 1.02 ± 0.04 3.45 ± 0.07 14.2 ± 0.40 2.88 ± 0.07 2.20 ± 0.11 2.96 ± 0.08 19.33 ± 0.88 18.2 ± 0.44
presence of calcium, aluminium, potassium, chlorides and sulphates. Fluorescence analysis of the powdered bark does not indicate presence of any fluorescent compound. Preliminary phytochemical screening
Physicochemical parameters
The results of determination of total ash, acidinsoluble ash, water-soluble ash, sulphated ash, loss on drying and different extractives are tabulated in Table 3. The qualitative analysis of ash indicated
Preliminary phytochemical screening indicated presence of alkaloids in chloroform, methanol and aqueous extracts while petroleum ether extract showed presence of steroidal moiety. Aqueous extract also gave positive tests for carbohydrates. Methanol,
216
INDIAN J NAT PROD RESOUR, JUNE 2011
Table 4 Description of TLC pattern of extracts of Persea macrantha bark Extract/S Visible pot No. Petroleum ether extract 1. -2. -3. -4. -5. 6. 7.
----
Chloroform extract 1. -2. -3. --
Under UV 254nm 366nm --------
----
After Spray
Light pink -Light brown -Purple -Dark brown White fluorescent -Blackish -Purple -Blackish brown --Yellow fluorescent
Pink Pink Pink
Rf
0.16 0.25 0.46 0.53 0.63 0.78 0.88
0.4 0.5 0.84
Table 5 Standards of Persea macrantha bark Parameter Total phenolics Total tannins Total carbohydrates Total alkaloids Mucilage content Unsaponifiable matter Crude fibre content a Mean value of three readings.
Plate 4 Powder characteristics of Persea macrantha bark: (a) fibres, (b) stone cells, (c) cork cells.
ethyl acetate and aqueous extract indicated presence of phenolic compounds. The TLC profile of petroleum ether extract indicated presence of seven compounds with the solvent system petroleum ether: ethyl acetate (8:2). Chloroform extract showed presence of four
Percentage (Meana ± SEM) 2.01 ± 0.11 0.11 ± 0.05 6.02 ± 0.30 4.20 ± 0.32 15.2 ± 0.42 19.66 ± 0.88 62.1 ± 0.63
compounds with the solvent system chloroform: methanol (8:1) (Table 4). The results of estimations of total phenolics, total tannins, total carbohydrates, total alkaloids, mucilage content, unsaponifiable matter and crude fibre content are presented in Table 5. Evaluation of physicochemical parameters is an indispensable part in preparation of modern monograph. Thus, different ash values, extractive values, loss on drying and fluorescence characteristics can be used for standardizing the crude drug samples. The TLC developed profiles can be used for identification as well as guide for isolation of various compounds from the extracts. Conclusion The diagnostic features have been established to identify P. macrantha bark. Some of the important diagnostic features of the bark are its characteristic odour, presence of ‘U’ shaped sclereids and numerous mucilage cells. The bark has shown remarkable
KULKARNI et al: PHARMACOGNOSY OF PERSEA MACRANTHA BARK
amount of alkaloids and polyphenolics. An important observation from phytochemistry point of view is absence of flavonoids and glycosides in the bark. The present studies on the bark of P. macrantha will be useful for its identification and authentication. References 1
The Wealth of India: A Dictionary of Indian Raw Materials and Industrial ProductsRaw Materials Series, Publication and Information Directorate, CSIR, New Delhi, India, 1962, Vol. VI, p. 204. 2 Kirtikar KR and Basu BD, Indian Medicinal Plants, Vol. III, International Book Distributors, Dehra Dun, Reprint Edn, 1999, pp. 2155-2156. 3 Nadkarni KM, Indian Materia Medica, Vol. I, Popular Prakashan, Mumbai, 2000, p. 759. 4 Gaind KN and Baveja SK, Investigation of Machilus macrantha Nees. I- Pharmacognostical and phytochemical study of root, J Am Pharm Assoc Sci Edu, 1960, 49, 659-662. 5 Tomita M, Gaind KN and Baveja SK, Structure of machiline: an alkaloid of Machilus macrantha, Yakugaku Zasshi, 1963, 83, 218. 6 Baveja SK, Garg ML and Joneja MP, Investigation of Machilus macrantha Nees. Part III, Indian J Pharm, 1968, 30, 11-13. 7 Gaind KN and Baveja SK, Investigation of Machilus macrantha Nees. II- Pharmacological action and chemical constitution of machiline, J Am Pharm Assoc Sci Ed, 1960, 49, 663-665. 8 Talapatra B, Ray T and Talapatra S, Structure and partial syntheses of Machicendiol, Machicenonol and MachiceninThree new nor-lignans from Machilus glucescens: Preferred confirmation of the side chain of Machicendiol, J Indian Chem Soc, 1978, 55, 1204-1208. 9 Talapatra B, Goswami S, Ghosh A and Talapatra SK, Alkaloids of Machilus glaucescens Wight: Machigline, A new phenolic oxoaporphine alkaloid, J Indian Chem Soc, 1982, 59, 1364-1368. 10 Hatapakki BC and Tatiya AU, Antiinflammatory activity of Machilus macrantha bark, Indian J Pharm Sci, 2003, 3, 240242. 11 Kulkarni YA, Gokhale SB, Veeranjaneyulu A, Surana SJ and Tatiya AU, Effect of Persea macrantha against acute inflammation and adjuvant-induced arthritis in Rats, Pharm Biol, 2009, 47(4), 304-308.
217
12 Kulkarni YA, Yele SU, Surana SJ and Gokhale SB, Pharmacognostical studies of Machilus macrantha leaves, Pharmacog Mag, 2008, 4(13), 83-88. 13 WHO (World health organization), Quality control methods for medicinal plants, AITBS publishers and distributors, Delhi, 2002, pp. 10-11. 14 Brain KR and Turner T, The Practical Evaluation of Phytopharmaceuticals, Wright-Scientechnica, Bristol, 1975, pp.10-12. 15 Reddy YSR, Venkatesh S and Ravichandra T, Pharmacognostical studies on Wrightia tinctoria bark, Pharm Biol, 1999, 37, 291-295. 16 Kay LA, The microscopic studies of drugs, Bailliere Tindall and Cox, London, 1938, pp. 77-96. 17 Pharmacopoeia of India, Vol. II, Govt. of India, Ministry of Health, Controller of Publication, New Delhi, 1996, p. A-52, A-54. 18 Pharmacopoeia of India, Govt. of India, Ministry of Health, Manager of Publications, New Delhi, 1966, pp. 390-394. 19 Wallis TE, Practical Pharmacognosy, J and A Churchill Ltd., London, 1953, p. 139. 20 Kokoski J, Kokoski R and Salma FJ, Fluorescence of powdered vegetable drugs under ultraviolet radiation, J Am Pharm Ass, 1958, 47, 715-717. 21 Pratt RJ and Chase CR, Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification, J Am Pharm Ass, 1949, 38, 324-333. 22 Badami SS, Gupta M and Suresh B, Pharmacognostical evaluation of Grewia tiliaefolia bark, Indian J Nat Prod, 2002, 18, 6-11. 23 Kokate CK, Purohit AP and Gokhale SB, Pharmacognosy, 30th Edn, Nirali Prakashan, Pune, 2004, pp. 593-597. 24 Harborne JB, Phytochemical Methods, 3rd Edn, Chapman and Hall, London, 1998, pp. 90, 203. 25 Singleton VL and Rossi JA, Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, Am J Enol Viticult, 1965, 16, 144-158. 26 Sadashivam S and Manickam A, Biochemical methods, New Age International Publishers, New Delhi, 1997, pp. 193-196. 27 Dubois M, Gilles KA, Hamilton JK and Rebers PA, Colorimetric method for determination of sugars and related substances, Anal Chem, 1956, 28, 350-356. 28 Kokate CK, Practical Pharmacognosy, Vallabh Prakashan, New Delhi, 1994, pp. 115-121. 29 Raghuramulu N, Madhavan K and Kalyanasundaram S, A manual of laboratory techniques, National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, 2003, p. 57.
