26 Phcog J | Dec 2012 | Vol 4 | Issue 34

O R I G I N A L A R T I C L EP H C O G J

ABSTRACT

Introduction: Substitution of Cinnamomum tamala (tamalapatra) with similar looking leaves of other Cinnamomum species is frequent among the people who use this spice in their food. On account of characteristic flavor, leaves of Cinnamomum verum are in use as spice among the indigenous people living throughout Western Ghats. To differentiate and to establish the identity of any article of plant origin, a thorough chemical fingerprint profile is vital. This investigation is an attempt to fingerprint the chemical properties and composition of a Cinnamon derived leaf spice – C. verum. Methods: Leaves from plants of C. verum growing wildly in South Canara district were collected and subjected to physico-chemical, HPTLC and GC-MS analysis. Results: Physico-chemical standards, HPTLC fingerprint and essential oil composition of wild C. verum used as spice were derived. While physico-chemical constants will serve the purpose of standardisation, volatile oil composition will be a diagnostic test for the identification and differentiation of leaves of C. verum from C. tamala. Conclusion: Chemical fingerprint profile of C. verum has been established for the identification and differentiation of this species from tamalapatra.

Keywords: C. zeylanicum, GC-MS, HPTLC, spice, tamalapatra.

Chemoprofile of tvakpatra; leaves of Cinnamomum verum J.S. Presl

Koppala Narayana Sunil Kumar,1* Billadi Sangeetha,1 Maheshwari Rajalekshmi,1 Basavaiah Ravishankar,1 Raghuveera Muralidhar2 and Betikeri Yashovarma3

1SDM Centre for Research in Ayurveda and Allied Sciences, Laxminarayana Nagar, Kuthpady, Udupi, INDIA 574 1182SDM Ayurveda Pharmacy, Laxminarayana Nagar, Kuthpady, Udupi, INDIA 574 118

3SDM College (Autonomous), Belthangady Tq., Ujire, Dakshina Kannada, INDIA 574 240

Submission Date: 10-8-2012; Review completed: 17-8-2012

INTRODUCTION

Substitution is a major concern in establishing the identity and quality of many herbal materials.[1] Usage of locally available plants as substitutes to their official source is a practice ubiquitous among the people who use plants for food and medicine. Many such herbs, even if inferior in quality, are picked up by indigenous people for day to day usage as drugs or spice. Cinnamomum is a genus of the fam-ily Lauraceae having many examples related to the issue of adulteration/substitution with closely knit species.[2] The similarity in macro-microscopical and flavor char-acteristics make different parts of members of genus Cinnamomum more prone to issues of substitution to

each other. Use of leaves of different species of Cinna-momum as a substitute to bay leaves or tamalapatra is a fact reported earlier,[3,4] similarly bark of different species of Cinnamomum are used as Cinnamon of commerce. Bark of C. verum (Syn. C. zeylanicum) – the Ceylon cinnamon or tvak in Ayurveda is an important medicinal drug as well as a spice used to flavor edibles throughout the world. In Ayurvedic therapeutics the botanical source of tvak is equated to bark of C. verum, but tvakpatra to leaf of C. tamala. The flavor characteristics of leaves of C. verum resemble to that of bark of C. verum and in turn to leaf of C. tamala. Survey of the local food traditions of South Canara district of Karnataka and Kasaragod district of Kerala revealed that leaves of C. verum are used widely to spice up different dishes. On account of similarity in flavor as well as easy availability, indigenous people use leaf of C. verum as an inexpensive substitute for commercial Cinnamon derived spices. As tvak patra or tamalapatra is an important com-modity in Ayurvedic medicine,[5] it was thought worth to undertake this investigation on the closely allied species. This study will serve as a preliminary step in the explora-tion of C. verum as a substitute for tvakpatra.

*Corresponding author. SDM Centre for Research in Ayurveda and Allied Sciences Laxminarayana Nagar, Kuthpady, Udupi, INDIA 574 118Phone: +91-820-2533971

E-mail: sunilkumarnarayanan@gmail.com

DOI: 10.5530/pj.2012.34.5

Koppala Narayana Sunil Kumar, et al.: Chemoprofile of tvakpatra; leaves of Cinnamomum verum J.S. Presl

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MATERIALS AND METHODS

Plant materials

Fresh flowering twigs of Cinnamomum verum were collected from plants growing wild in Kasaragod district and identi-fied using literature available in different floras.[6,7] Leaves from botanically identified twigs were harvested and shade dried. Air dried leaves were powdered and stored in air tight containers for further chemical examination.

Instrumentation and techniques

Physico-chemical properties were determined as per the WHO guidelines. Volatile oil of the sample of C. verum were distilled using Clavenger’s apparatus.[8] TLC charac-terization of the n-hexane extract was done as per stan-dard procedure.[9] Ten and twenty µl of the extracts were applied on a precoated silica gel F254 aluminum plates to a band width of 8 mm using Linomat 5 TLC applicator. The plate was developed in toluene: ethyl acetate (7:1) and the developed plates were visualized and scanned under UV 254, 366, under white light and after derivati-sation in vanillin-sulphuric acid spray reagent at 620 nm. Rf, colour of the spots, densitometric scan and superim-posability of densitogram were recorded.[10] GC-MS of essential oil of C. verum was carried out using Shimadzu gas chromatograph with a SE-30 10% Chromosorb-W packed stainless steel column (2 m × 2 mm). Oven pro-gramme: 60°C (5 min), 60°–260°C (5°C/min), 260°C (10 min); carrier gas – nitrogen, flow rate 40 ml/min; injector temperature 240°C; detector temperature 240°C. Individual components were identified by database of mass spectra matching with literature available in the digital libraries like NIST and WILEY by comparison of their RT values.

RESULTS

Results obtained for the physico-chemical tests are tabulated in Table 1. Rf values of the spots and their colour on TLC of n-hexane extract is tabulated in Table 2. TLC photodocumentation of n-hexane extract is shown in Figure 1. HPTLC Densitometric scan of n-hexane extract of C. verum at UV 254, 366 nm, under white light, and after derivatisation with vanillin-sulphuric acid at 620 nm are shown in Figure 2. The GLC chromatogram for the essential oil of C. verum is presented in Figure 3. Compounds detected by the GC-MS of the essential oil C. verum, the respective RT values and the percentages of the compounds detected are tabulated in Table 3.

DISCUSSION

Due to wild occurrence in Western Ghats, morpho-logical similarity, and characteristic fragrance, leaves of C. verum has been used commonly by the people for flavouring various edibles. With this background infor-mation it was decided to derive chemical fingerprint to

Table 1. Physico-chemical parameters Cinnamomum verum.

Parameters ValueLoss on drying at 105°C % w/w 8.21pH of water soluble extractive 6.27Total ash % w/w 4.94Acid insoluble ash % w/w 0.360n-Hexane soluble extractiveAlcohol soluble extractive % w/w

13.96 9.96

Water soluble extractive % w/w 7.45Swelling factor ml 13

Table 2. Rf value of n-hexane extract of Cinnamomum verum 20 µl.At UV 254 nm At UV 366 nm Under White light Post-derivatisation0.04 Green 0.04 F violet – –

– – 0.13 Yellow 0.13 Brown0.19 Green 0.19 F blue – –

– 0.21 F brown – 0.21 Brown0.32 Green – – –

– – – 0.40 Purple0.46 Green 0.46 F pInk 0.46 Green 0.46 Violet

– 0.52 F blue – –0.55 Green 0.55 F pink 0.55 Yellow 0.55 Blue

– – – –0.61 D green 0.61 F pink 0.61 Green 0.61 Green0.70 D Green – – –0.75 Green – – 0.75 Pink0.90 Green 0.90 F blue – 0.90 L greenD – dark; F – fluorescent; L – light

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28 Phcog J | Dec 2012 | Vol 4 | Issue 34

establish the identity of this less known spice. Physico-chemical, HPTLC and GCMS fingerprint was obtained for the leaf in the present study.

Results obtained for physico-chemical parameters such as loss on drying, pH of water soluble extrac-tive, total ash, acid insoluble ash, n-hexane soluble extractive, alcohol soluble extractive, water soluble extractive and swelling factor recorded can be used as standardisation parameter for routine identification of C. verum leaves.

Figure 1. TLC photodocumentation of n-hexane extract of Cinnamomum verum.

Figure 2. HPTLC Densitometric scan of n-hexane extract of Cinnamomum verum.

Figure 3. GLC chromatogram for the essential oil of Cinnamo-mum verum.

Koppala Narayana Sunil Kumar, et al.: Chemoprofile of tvakpatra; leaves of Cinnamomum verum J.S. Presl

Phcog J | Dec 2012 | Vol 4 | Issue 34 29

HPTLC fingerprinting revealed the presence of various phytoconstituents with their respective Rf values. The photo documentation of the plates showed numerous bands under UV 254, 366, white light, and after derivati-sation with vanillin sulphuric acid reagent. On photo doc-umentation under 254 nm there were 9 spots (Figure 1.1), 8 spots under 366 nm (Figure 1.2), 4 spots under white light (Figure 1.3) and 8 spots after derivatisation with van-illin sulphuric acid (Figure 1.4). Densitometric scan at 254 nm revealed 13 peaks corresponding to 13 compounds in the n-hexane extract in the solvent system used for the sep-aration, compounds with Rf 0.71 (36.5%), 0.91(17.32%) were major ones (Figure 2.1). Densitometric scan at 366 nm showed 13 peaks, compounds with Rf 0.45 (24.19%), 0.65 (28.00%) were major ones (Figure 2.2). Densitometric scan at 540 nm showed 4 peaks, compounds with Rf 0.62 (69.44%), 0.46 (18.96%) were major ones (Figure 2.3) and densitometric scan at 620 nm showed 11, compounds with 0.42 (23.57%), 0.58 (23.00%) were the major peaks (Figure 2.4) detected.

GC-MS of volatile oil from the leaves of C. verum revealed 24 volatile constituents (Figure 3 and Table 3). The major compounds detected were eugenol (81.60%), linalool (5.83%), β-caryophyllene (4.50%) and benzyl

benzoate (1.95%). Present compositional analysis of the essential oil revealed all constituents reported from leaves of C. verum by earlier workers[11] other than γ-elemene and germacrene D. γ-Elemene was reported from spe-cies of Cinnamomum namely C. cordatum, C. porrectum, C. burmanii and C. cassia. Germacrene D was reported from C. angustifolium and C. verum (fruit and flower).[12] Presence of γ-elemene and germacrene D is the first time report from the leaves of C. verum growing wild.

Compounds such as eugenol, linalool, α-pinene, camphene, β-pinene, benzaldehyde, myrcene, limo-nene, p-cymene, benzyl acetate, α-terpineol, cin-namaldehyde, geraniol, linalool acetate and benzyl cinnamate were reported from the volatile oil of leaves of C. tamala by other workers earlier.[13-15] From the pres-ent study, occurrence of 10 common compounds such as α-pinene, camphene, benzaldehyde, β-pinene, p-cymene, limonene, linalool, cinnamaldehyde, euge-nol and benzyl benzoate in both C. tamala and C. verum were confirmed. Exhaustive pharmacological evalua-tion might be conclusive to designate leaves of C. verum as a substitute or as an adulterant for C. tamala. The present study has concluded chemical identity tests for the detection of this species.

Table 3. Compounds detected by GC-MS of leaf essential oil from Cinnamomum sp.Wild C. verum

Sl No. Compounds RT Area % C. verum10 C. tamala12-14

1 α-Pinene 9.167 0.62 + +2 Camphene 9.973 0.29 + +3 Benzaldehyde 10.899 0.13 + +4 β-Pinene 11.108 0.31 + +5 α-phellandrene 12.165 0.18 + –6 Δ3-Carene 12.583 0.09 + –7 p-Cymene 13. 014 0.13 + +8 Limonene 13.158 0.22 + +9 β-Phellandrene 13.365 0.43 + –

10 Linalool 16.242 5.83 + +11 γ -Terpinene 16.242 0.08 + –12 α-Terpinene 20.617 0.42 + –13 Cinnamaldehyde 21.316 0.88 + +14 Eugenol 25.574 81.60 + +15 α-Copaene 29.166 0.10 + –16 β-Caryophyllene 29.500 4.50 + –17 Cinnamyl alcohol 31.654 0.29 + –18 α-Humulene 32.747 0.73 + –19 Germacrene D 33.234 0.08 – –20 γ -Elemene 34.258 0.08 – –21 δ-Cadinene 34.875 0.19 + –22 Spathulenol 35.529 0.17 + –23 Caryophyllene oxide 38.483 0.70 + –24 Benzyl benzoate 38.815 1.95 + +

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ACKNOWLEDGEMENTS

Authors are highly grateful to Dr. D. Veerendra Heggade, revered President of SDM Educational Society for the guidance and support. Help rendered by Dr. Anamik Shah and Pankaj Kachhadia, Saurashtra University, Rajkot, towards GC-MS analysis is also gratefully acknowledged.

REFERENCES

1. Garg S. Substitute and Adulterant Plants. New Delhi: Periodical Experts Book Agency; 1992.

2. Baruah A, Nath SC, Boissya CL. Systematics and diversities of Cinnamomum species used as Tejpat spice in north-east India. J Econ Tax Bot. 2000; 24: 361–74.

3. Sunil Kumar KN. Pharmacognosical evaluation of Cinnamomum tamala (Buch. - Ham.) Nees and Eberm. (Tamalapatra) and few of its allied species. M.Sc. Thesis, Institute Ayurvedic Medicinal Plant Sciences, Gujarat Ayurved University, Jamnagar, Gujarat, 2006.

4. Koppala Narayana Sunil Kumar, Maheshwari Rajalekshmi, Billadi Sangeetha, Basavaiah Ravishankar, Raghuveera Muralidhar. Chemical examination of leaves of Cinnamomum malabatrum (Burm. f.) Blume sold as Tamalapatra. Phcog J 2012; 31: 11–15.

5. The Ayurvedic Pharmacopoeia of India. New Delhi: Ministry of Health and Family Welfare, Department of ISM & H, Govt. of India; 1999; Part-I, Vol. I: pp. 153–4.

6. Cooke TCIE. Flora of the Presidency of Bombay. London: Taylor & Francis; 1903; Vol. I: pp. 282–3.

7. Gamble JS. Flora of the Presidency of Madras. Calcutta: Botanical survey of India; 1925; (Vols. II).

8. Quality control methods for medicinal plant materials. Geneva: WHO; 1998; pp. 16–27.

9. Stahl I. Thin layer chromatography, A Laboratory Hand Book (student edition). Berlin: Springer-Verlag; 1969; pp. 52–86, 127–8.

10. Sethi PD. High Performance Thin Layer Chromatography (1st Edition). New Delhi: CBS Publishers and Distributors; 1996; Vol X: pp. 1–56.

11. Senanayake UM, Wijesekera ROB. Chemistry of Cinnamon and Cassia. In: Ravindran PN, Nirmal Babu K, Shylaja M. Cinnamon and Cassia – The genus Cinnamomum. Washington DC: CRC Press, 2004: 80–120.

12. Ravindran PN, Nirmal Babu K, Shylaja M. Cinnamon and Cassia – The genus Cinnamomum. Washington DC: CRC Press, 2004.

13. Nath SC, Hazarika AK, Singh RS. Volatiles oil of leaves of Cinnamomum tamala Nees & Eberm. From North East India. J Spices & Aromatic Crops. 1994:33–5.

14. Gulati BC, Agarwal SG, Thappa RK, Dhar KI. Essential oil of tejpat (Kumaon) from Cinnamomum tamala. Indian Perfum. 1977; 21: 15–20.

15. Sood RP, Padha CD, Talwar YP, Jamwal RK, Chopra MM, Rao PR. Essential oils from the leaves of Cinnamomum tamala Nees and Eberm growing in Himachal Pradesh. Indian Perfum. 1979; 23: 75–8.