Pharmacognostical and phytochemical evaluation of ... - NOPR

Indian Journal of Natural Products and Resources Vol. 3(1), March 2012, pp. 33-39

Pharmacognostical and phytochemical evaluation of Cinnamomum wightii Meissn. flowers D Laloo1, A N Sahu1*, S Hemalatha1 and S D Dubey2 1

Pharmacognosy Research Laboratory, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi- 221 005, Uttar Pradesh, India 2 Department of Dravyaguna, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi- 221 005 Received 17 January 2011; Accepted 3 October 2011

Cinnamomum wightii Meissn. (Family ⎯ Lauraceae) is a tree mainly found in the Western Ghats and Shola forest of the South India. Pharmacognostical and phytochemical evaluation of crude drug has been done as per WHO guidelines and IHP 2002. The crude flower buds are globoid to round in shape, dark brown with 1-2 cm long pedicel. Transverse section through petals showed both the outer and inner epidermis with numerous stone cells and secretory glands scattered in the cortex. The quantitative values found are: foreign organic matter (0.71%), loss on drying (11.88%), total ash (6.41%), acid insoluble ash (2.07%), water soluble ash (0.38%), alcohol soluble extractive (28.63%) and water soluble extractive (7.23%), foaming index (200), swelling index (0.85 ml), volatile oil content (0.20%) and pesticide residue content. The powdered crude flower buds have shown the presence of stone cells, vessel element, covering trichomes, spiral xylem vessel and fragments of anthers filamentous cell layer. Phytochemical screening of methanolic extract showed the presence of alkaloids, glycosides, flavonoids, saponins, coumarins, reducing sugars, phenolics, tannins, sterols and mucilage. Quantitative estimation of the methanolic extracts of various phytoconstituents, viz. total tannins (156.5 mg/g), total phenolics (146.40 mg/g), total flavonoids (30 mg/g) and total flavonols (3.6 mg/g) content has been estimated spectrophotometrically. These findings may serve as diagnostic tools for identification of the crude drug. Keywords: Cinnamomum wightii, Fluorescence analysis, Nagakeshara, Pesticide, Phenolics, Quality control, Standardization, Thin layer chromatography. IPC code; Int. cl. (2011.01) ⎯ A61K 36/00

Introduction India has a rich cultural heritage of traditional medicines which widely includes Ayurveda, Homoeopathy and Siddha system of medicines. Plants are the basic source of medicines in all these systems. The commerce of medicinal plants is flourishing globally day by day and besides there is also a huge demand of plant drugs all over the world, thus there might always be a propensity for adulteration and substitution of the crude drugs with the genuine ones. Hence, standardization of medicinal plants and natural products are utmost need of the time which till now is least attended. According to World Health Organization the standardization of any medicinal plants can be done as per the methods proposed on various parameters. Cinnamomum is the most popular spice used these days. The genus Cinnamomum Blume (Family ⎯ ——————— *Correspondent author: E-mail: [email protected]; Phone: +91 5426702714

Lauraceae) comprises several species, which occur in Asia and Australia. These are evergreen trees and shrubs and most of the species are aromatic. All the Cinnamomum species were used to treat multiple disorders. Trees of C. wightii Meissn. (CW) are found mainly in the Shola forest of South India and also in the Western Ghats. The flower buds are used as one of the ingredients in the preparation of Siddha medicines mainly in the formulation of Amukkarac curanam1. The bark, leaves and oil obtained from the plant are used to treat paralytic disorders, deficiency in digestive power, abdominal disorders, cough, dysuria and gynaecological disorders2. Tender fruits, including style and calyx are astringent, aromatic, stimulant and carminative. In addition to this, it is also used to treat wounds, fever, intestinal worms, headache and menstrual problems3. The main aim of this study is to develop a standardization method for this plant both pharmacognostically and phytochemically. Since this drug is commercially sold in the Indian market under the name Nagakeshara, the

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INDIAN J NAT PROD RESOUR, MARCH 2012

purpose of our investigation is to aid its importance in the field of research and as well as its commercial gradient where it might be possibly adulterated with other marketed drugs like Mesua ferrea Linn., Ochrocarpus longifolius Benth. & Hook. (Rattan Nagakeshara), Calophyllum inophyllum Linn. and Dillenia pentagyna Roxb. (Malabar Nagkesara)4. Materials and Methods

for medicinal plants material and from other official methods9. The preparation of individual solvent extract was also done by cold maceration process by dissolving the powdered drug in hexane, petroleum ether, benzene, chloroform, ethyl acetate, ethanol and aqueous. The results were calculated in terms of percentage yield weight by weight of air-dried plant material. Fluorescence analysis of the crude powder drug was also carried out as per the standard methods10.

Chemicals and Instruments

Phytochemical evaluation and thin layer chromatography

All chemicals, solvents and reagents used were of standard analytical grade. Photomicrographs in different magnifications of all necessary cells and tissues were taken using microscopic digital camera (Labcron). Pre-coated aluminium silica gel plates 60 F254 (Merck, Germany) and Clevenger apparatus for volatile oil determination and UV cabinet were used for the study.

The preliminary phytochemical screening of the methanolic extracts obtained by Soxhlet extraction was carried out for the detection of various phytoconstituents5,11. Preparation of extracts, thin layer chromatography and developing of chromatogram was done based on the standard methods12. The TLC plates used for the stationary phase are the pre-coated aluminium silica gel plates 60 F254. The mobile phase for the developing of the chromatograms contains the mixture of solvents having varying polarities. For identification of the different classes of phytoconstituents spraying reagent used were: 5% ferric chloride (for phenolics), 2% ethanolic aluminium chloride (for flavonoids), Liebermann-burchard reagent (for triterpenes and steroids), Dragendorff reagent (for alkaloids) and benzidine sodium metaperiodate reagent (for glycosides). Quantitative estimation of various phytoconstituents, viz. total phenolic, flavonoid and flavonol was determined as per the methods of Kumaran et al13, whereas the total tannin content was estimated following the methods of Grubesic et al14. All the results for the quantitative estimation were carried out in triplicates and were expressed as the Mean ± S.E.M (Standard Error Mean).

Plant material and extraction process

The commercially available C. wightii crude flower buds were procured from Chennai, Tamil Nadu, India. The specimen was identified by Prof. S. D. Dubey, Department of Dravyaguna, Faculty of Ayurveda, Institute of Medical Science, Banaras Hindu University, Varanasi, India. A voucher specimen (COG/CW/015) has been deposited for future references in the Pharmacognosy Research Laboratory, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi. The coarse powder drug (250 g) was extracted with methanol and the residue was concentrated under rotary evaporator and finally dried and store in desiccator. The extractive value of the dried extract was found to be 30.55% w/w of the dried plant material. Macroscopic and Microscopic evaluation

The macroscopical and microscopical study was done based on the standard methods5,6. Moreover for the microscopical studies, cross sections were prepared and stained properly as per the usual methods7,8. Physico-chemical evaluation

The determination of physico-chemical constants, viz. percentage of foreign matter, loss on drying, ash values (total ash, acid insoluble ash and water soluble), swelling index, foaming index, volatile oil content and pesticide residue was done as per WHO 2002 guidelines on quality control methods

Results and Discussion Macroscopical characters

The crude flower samples were globose to round in shape, dark brown to black in colour when dried. The pedicles were short with 1-2 cm in length. Petals (1-3 cm) during the bud stage were seemed to be attached with each other to form a crown that was fixed to the thalamus (Plate 1). Microscopical characters Specimen sections

The transverse section through the flower petal was taken and stained with a mixture of phloroglucinol

LALOO et al: QUALITY CONTROL PARAMETERS OF CINNAMOMUM WIGHTII MEISSN. FLOWERS

and HCl (1:1) as represented in the Plate 2. The histological section indicates the presence of both upper and lower epidermis, secretory cell layer, stone cell layer, epidermis and a layer of anthers cells. The upper epidermis is uniform, laterally elongated composed of an outer cuticle and shows the absence of stomata. Cortical region is made up of thin walled parenchymatous cells and embedded with numerous stone cells and secretory glands. Endodermis is well developed and followed by a layer of anther thus bearing the epipetalous nature of a flower. Powder characteristics

The powder of the C. wightii flower buds appeared greyish to dark brown in colour with a slight aromatic

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odour and bitter in taste. The powder drug was visualized and studied under a digital microscope having magnifications (10x × 10x) and (10x × 45x) and the staining was done with the mixture of phloroglucinol and HCl in the ratio 1:1. Lignified xylem vessel and clusters of stone cells or sclerenchymatous cells were clearly visible in the powder drug and stained pink when treated with the staining reagent. Stone cells present are numerous and appeared to be polygonal in shape. Numerous unicellular covering trichomes were also found to be present in the powder drug. Xylem vessels found were mostly elongated and contains spiral thickening. Layers of filamentous cells were also found to be present in the powdered drug as shown in the Plate 3. Physico-chemical evaluation Quantitative values

Plate 1—Crude drug of Cinnamomum wightii (Meissn.) flower buds

The result obtained for the physicochemical constants was shown in the Table 1. The percentage of foreign matter was found to be less in the crude flower buds which imply that it is almost free from any other plant part or any other matter other than the crude drug itself. The percentage of loss on drying is quite high which indicates that the crude drug might contains an extensive amount of moisture. The total ash value (6.41%) present in the crude drug indicates the presence of both organic matter (physiological) and inorganic matter (non-physiological). Acid

Plate 2—Transverse section of Cinnamomum wightii (Meissn.) through petals

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Plate 3—Microscopical powdered drug characters of Cinnamomum wightii. [VE- Vessel element, SC- stone cell, TR- trichome, FCL- filamentous cell layer, XV- xylem vessel] Table 1—Physicochemical constants of Cinnamomum wightii flower buds S No. Parameters Results 1 Foreign matter 0.71 % (w/w) 2 Loss on drying 11.88 % (w/w) 3 Total ash 6.41 % (w/w) Acid Insoluble ash 2.70 % (w/w) Water soluble ash 0.38 % (w/w) 4 Alcohol soluble extractive 28.63 % (w/w) Water soluble extractive 7.23 % (w/w) 5 Foaming Index 500 6 Swelling Index 0.85 7 Volatile oil determination 0.20 % 8 Chlorinated pesticide residue (mg/kg) 0.0070 TS1 (First elute) 0.0150 TS2 (Second elute) Phosphated pesticide residue (mg/kg) 0.0014 TS1 (First elute) TS2(Second elute) 0.0012 TS3 (Third elute) -

insoluble ash measures the amount of silica presence especially in the form of sand or siliceous earth material whereas water soluble ash is used to detect the presence of material exhausted by water15,16. The crude drug was also found to contain a high foaming and swelling index which may be suggested due to the

presence of saponins and mucilages. Moreover, there was a negligible amount of volatile oil present in the crude drug. The pesticide residue was also determined as per WHO guidelines and the different fractions of the pesticides was obtained from the column packed with florisil F254 as a stationary phase and eluted with mobile phase containing different mixtures of diethyl ether and light petroleum ether. Two fractions for chlorinated pesticide (TS1 and TS2) and three fractions for phosphate pesticide (TS1, TS3 and TS3) were collected and their values were shown in the Table 1. Certain official books like Quality control methods for medicinal plant materials, WHO, Geneva has provided some information about the limits of pesticides in which the phosphated pesticide having the content in between 0.05-0.1 mg/kg of the plant material is considered to be of no contamination8. So from this we can conclude that C. wightii flower bud was shown to have no contamination with phosphate pesticides. Fluorescence powder drug analysis

As it is known that when a powder drug is made into contact with certain chemical reagent (either acid


or bases) it gives a colourful fluorescence tinge. The phytoconstituents present in the crude drug interact with the chemical reagents and may produce certain product which may be present inside the cell or may come out of the cell and react in the medium, thus resulting in a specific fluorescence pattern. This is the basis of fluorescence analysis. The respective Table 2, specifies the results in which the powder drug of the C.wightii flower buds produces fluorescence in day light as well as under long UV light (365nm). Fluorescence analysis was carried out in an ultraviolet cabinet (UV cabinet R/340/OC). The identification of the colours was done using the standard colour index to achieve a better colour resolution17. Thus, it can be concluded that the fluorescence test gives a brief highlight of how powders of different natures can be compared and distinguished properly. Phytochemical evaluation and thin layer chromatography

Phytochemical screening of the methanolic extracts of C.wightii indicates the presence of some major constituents which are flavonoids, phenolics, tannins, steroids, glycosides, alkaloids and saponins whereas other phytoconstituents like volatile oil and reducing sugars were found to be minor. Proteins and amino acids were found to be absent in the extract. Mucilage was also found to be present which gives positive test when the powdered plant material was made into contact with water thus producing a thick sticky medium. The results for the cold macerated extractive value of the individual solvent extract were shown in the Table 3. The values were expressed in terms of percentage weight/weight of the dried plant material. The identification of major phytoconstituents in the various extracts of C. wightii by the use of TLC was

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also studied to further confirm the presence of the possible phytoconstituents and this was represented in the Plate 4. The solvent system for developing the chromatogram contains the mixture of chloroform and methanol (9:1). The TLC chromatogram for the different phytoconstituents can be identified clearly after spraying with their respective spraying reagent. Alkaloid indicates the presence of a light orange colour after spraying with Dragendorff’s reagent having an Rf of 0.18. Flavonoids give two bright yellow spots after spraying with alcoholic aluminium chloride with an Rf value of 0.27 and 0.43. Steroids (Rf 0.32) gives purplish colour when sprayed with Liebermann-burchard reagent. Phenolics (Rf 0.13) gives a bluish colour when sprayed with ferric chloride reagent. For glycosides detection a single cream to yellow colour spots (Rf 0.66) were seen after spraying with benzidine sodium metaperiodate reagent. Table 4 represents the quantitative estimation of total phenolics, tannins, flavonoids and flavonol components present in the methanolic extracts of C. wightii. Literatures revealed that phenolics and flavonoids are the two main phytoconstituents that are mainly responsible for the antioxidative property18. These compounds possess a broad spectrum of chemical and biological activities including radical Table 3—Percentage yield of crude flower buds of Cinnamomum wightii extract from various solvents S No.

Solvent used

1. 2. 3. 4. 5.

Hexane Petroleum ether (40-60°C) Benzene Chloroform Ethyl acetate

Percentage yield 12.60 8.80 30.00 33.60 38.80

Table 2—Fluorescence powder drug analysis of Cinnamomum wightii Meissn. S No.

Powder + Reagent

Fluorescence in daylight

Fluorescence under UV

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Powder as such Powder + 1N NaOH in methanol Powder + 1N NaoH in water Powder + 1N HCl in methanol Powder + 1N HCl in water Powder + 1N HNO3 in methanol Powder + 1N HNO3 in water Powder + Iodine (5%) Powder + FeCl3 (5%) Powder + KOH (50%) Powder + Ammonia (25%) Powder + Picric acid (saturated) Powder + Acetic acid

Dark golden rod (184,134,11) (B8860B) Orange (255,165,0) (FFA500) Dark red (139,0,0) (8B0000) Brown (165,42,42) (A52A2A) Golden rod (218,165,32) (DAA520) Brown (165,42,42) (A52A2A) Gold (255,215,0) (FFD700) Golden rod (218,165,32) (DAA520) Dark golden rod (184,134,11) (B8860B) Dark red (139,0,0) (8B0000) Brown (165,42,42) (A52A2A) Yellow (255,255,0) (FFFF00) Dark golden Rod (184,134,11) (B8860B)

NF Dark khaki NF NF Pale green (152,251,152) (98FB98) NF NF Pale green (152,251,152) (98FB98) NF Pale Green (152,251,152) (98FB98) Yellow green (154,205,50) (9ACD32) NF Medium aquamarine (102,205,170) (66CDAA)

NF: No fluorescence


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Plate 4—Thin layer chromatography of various phytoconstituents of Cinnamomum wightii [a- Phenolics; b- Triterpenoids/steroids; c- Flavonoids; d- Alkaloids; e- Glycosides] Table 4—Percentage yield, total amount of phenolic, tannin, flavonoid and flavonol content of the methanolic extract of Cinnamomum wightii. [Mean ± S.E.M] Sample

C.wightii

% Yield (Methanolic extract)

Total phenolic (mg/g) plant extract (in GAE*)

Total tannin (mg/g) plantextract (in TAE*)

Total flavonoid (mg/g) plant extract (in RE*)

Total flavonol (mg/g) plant extract (in RE*)

28.63

156.6 ± 2.85

146.4 ± 1.04

30 ± 0.52

3.60 ±0.25

GAE*- Gallic acid equivalent; TAE*- Tannic acid equivalent; RE*- Rutin equivalent

scavenging properties. Thus the extracts of C. wightii can acts as a potential candidate for antioxidative property due to its high phenolic content and which can be further investigated using various antioxidant models. Conclusion The present study on pharmacognostical and phytochemical evaluation of C. wightii will be providing useful information with regards to its correct identity and will helps to differentiate from other closely related species as well as from other commercially available crude drugs sold in the market under the name Cinnamomum. Acknowledgements The University Grant Commission (UGC) is highly acknowledged for their financial assistance during the tenure of our work. References 1

Brindha P, Saraswathy A, Mageswarai S and Vijayalakshmi R, Powder microscopy of amukkarac curanam, Bull MedicoEthno-Bot Res, 2004, 25(1-4), 47-59.

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Pullaiah T, Encycolpedia of World Medicinal Plants, Regency Publications, New Delhi, India, 2006, Vol. 2, p. 562. 3 Muthiah M and Bonfilius V, Ethnobotanical uses of Cinnamomum species, Tamil Nadu, India, Ethnobot. Leaflets, 2008, 12, 150-155. 4 Joseph C R, Ilanchezhian R, Patgiri Biswajoyti and Harish C R, Pharmacognostical study of Nagakeshara (Mesua ferrea Linn.) - An ingredient in Vyaghrihareetaki Avaleha, IJRAPInt J Res Ayurv Pharm, 2010, 1(2), 264-272. 5 Khandelwal K R, Practical Pharmacognosy, Techniques and Experiments, 17th Edn, Nirali Prakashan Publishers, Pune, India, 2007, pp. 9-22, 149-154. 6 Brain K R and Turner T D, The Practical Evaluation of Phytopharmaceuticals, Wright-Scientechnica, Bristol, 1975, pp. 36-45. 7 Johansen D A, Plant Microtechnique, McGraw Hill Book Co, New York, 1940, p. 182. 8 Quality control methods for medicinal plant materials, World Health Organization, Geneva, A.I.T.B.S. Publishers and Distributors, Delhi, 2002, pp. 8-60. 9 Indian Herbal Pharmacopoeia, Revised Edn, Published by Indian Drug Manufacturers, Mumbai, 2002, pp. 495-496. 10 Chase C R and Pratt R J, Fluorescence of Powdered Vegetable drugs with particular reference to Development of a System of Identification, J Am Pharmacol Assoc, 1949, 38, 32. 11 Kokate C K, Practical Pharmacognosy, 1st Edn, Vallabh Prakashan, New Delhi, India, 1986, pp. 15-30.


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Wallis T E, Text book of Pharmacognosy, 15th Edn, CBS Publishers, New Delhi, India, 1985, p. 561. 16 Sahu Alakh N, Hemalatha S, Sairam K, Laloo D and Patra A, Quality Control Studies of Ochrocarpus longifolius flower buds, Pharmaconosy J, 2010, 2 (6), 118-123. 17 http://en.wikipedia.org/wiki/Web_colors 18 Okawa M, Kinjo J, Nohara T and Ono M, DPPH (1, 1- Diphenyl-2-picrylhydrazyl) radical scavenging activity of flavonoid obtained from some medicinal plants, Biol Pharm Bull, 2001, 24, 1202-1205.

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