Cinnamomum Tamala (Buch.-Ham.) T.Nees & Eberm
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Indian Journal of Traditional Knowledge Vol. 13 (4), October 2014, pp. 691-697 Anti-cholinesterase potential of Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm. leaves Manoj K Dalai1, Santanu Bhadra1, Sushil K Chaudhary1, Arun Bandyopadhyay2 & Pulok K Mukherjee1* 1School of Natural Product Studies, Jadavpur University, Kolkata 700 032, India; 2CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India E-mail: [email protected] Received 07.10.13, revised 25.11.13 Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm. (Lauraceae) leaves are well known as bay leaves which are popular for its aroma. A part from its extensive culinary uses, this spice has several uses in traditional practice for treatment of rheumatism, immunomodulation and also used as brain tonic. The cinnamon oil locally known as Tejpat oil obtained from the bay leaves used in alcoholic beverages and confectionaries. The present investigation was aimed to screen the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of the extract of C. tamala. Standardization of the cinnamon oil of bay leaves was performed by HPLC method using linalool as biomarker. In vitro evaluation of anticholinesterase activity of bay leaves extract and oil was performed by TLC bio-autography and 96 well micro titer plate methods. HPLC analysis was confirmed the presence of linalool as an important phytoconstituents and its retention time was found to be 5.314 min. The outcome of the study demonstrated that the cinnamon oil obtained from leave of C. tamala possess maximum inhibition against AChE (IC50: 94.54 ± 0.774 µg/ml) and BChE (IC50: 135.56 ± 0.912 µg/ml) than the methanol extract. The result also explains that C. tamala is more sensitive to AChE enzyme than the BChE. Hence, C. tamala may be explored as anti-cholinesterase agent further for the better and safer management of Alzheimer's diseases. Keywords: Cinnamomum tamala, Acetylcholinesterase, Butyrylcholinesterase, Linalool, Spices IPC Int. Cl.8: A61K 36/00, CO1, CO7, A23L 1/22, C12N, A61K 38/43, C11C 3/00, BO3B 3/02 Cinnamomum tamala (family Lauraceae) is also Level of acetylcholine in the brain decreases in the known as Indian Cassia. The leaves are commonly Alzheimer’s disease (AD); which is principally called as bay leaves. For centuries, it is used as characterized by impaired memory and disturbed home culinary spices for its rich aroma. In behavior thus, AChE and BChE inhibitors are being traditional healing practices, bay leaves are used as developed for the symptomatic treatment of AD.The brain tonic, anthelminthic, diuretic and also used in results of previous studies at our laboratory indicated treatment of liver and spleen inflammation1. The that essential oils obtained from spices possess cinnamon oil of the bay leaves is locally known as significant AChE/BChE inhibitory activity6,7. The Tejpat oil is extensively used for flavoring foods current study was undertaken to evaluate its and formulation of liquors and confections3. This anticholinesterase potential of the standardize extract buff colored oil is used in wide range of diseases, of C. tamala so as to validate the neuroprotective viz. anti-flatulent, diuretic, hypoglycemic and activity of this spices. analgesic in dental preparations2. Reported GC–MS analysis of the oil showed the presence of Methodology 63 compounds in it, among them β-Caryophyllene, Collection and authentication of plant material linalool and caryophyllene oxide are the main The dried leaves (200 gm) of C. tamala was 4 constituents . Apart from this, therapeutic collected in the month of November 2011 from a phytochemicals compounds like, viz. α-pinene, commercial supplier of Kolkata, West Bengal, camphene, myrcene, limonene, 1, 8-cineole, India and authenticated. A voucher specimen of p-cymene, methyl eugenol and eugenol acetate are the plant material (SNPS 1259) has been deposited 5 also reported in oil . in the herbarium of School of Natural Products ___________ Studies, Jadavpur University, India for *Corresponding author further study. 692 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 4, OCTOBER 2014 Chemical and reagents performance liquid chromatography (RP-HPLC) 5, 5-dithiobis [2-nitrobenzoic acid] (DTNB), (Shimadzu Prominence, Kyoto, Japan) which Galantamine and Linalool was procured from Sigma equipped with Shimadzu LC-20 AD UFLC (Poole, UK). Acetylcholinesterase enzyme (AChE) reciprocating pumps with variable Shimadzu from bovine erythrocytes, butyrylcholinesterase SPD-M20A Prominence PDA detector. 20 µl of (BChE) from equine serum, acetyl thiocholine iodide cinnamon oil and standard linalool were used for (ATCI), butyrylthiocholine iodide (BTCI), methanol HPLC analysis at a concentration of 1 mg/ml. The and all other organic solvents (HPLC reagent grade) prepared sample was injected into 20 µl capacity of were purchased from Merck, India. Rheodyne loop injector. Standard linalool and cinnamon oil of C. tamala were analyzed on a Cold maceration Phenomenex-Luna (Torrance, CA, USA) C column Leaf of C. tamala was air dried in the shade and 18 (250×4.6 mm, 5 µ particle size) as stationary phase pulverized into coarsely powder in a mechanical and methanol: water (80:20 v/v) as mobile phase in grinder (Bajaj platini, India, PX 73 M, wattage: 550w, which 1 ml/min maintained the flow rate. During voltage: 230 v 50 Hz AC). The powdered plant elution the temperature of the column was maintained material (190 gm) was extracted by cold maceration at 55ºC and reading was monitored at 210 nm10. A with (750 ml) methanol. The powder was soaked in calibration curve was prepared by using range of methanol for 72 hrs with occasional stirring at room standards to quantify the percentage of linalool temperature. After 72 hrs, the same procedure was present in oil. The chromatographic peak followed thrice for maximum extraction from the corresponding to cinnamon oil was identified by powder. All the extracted solvent then pooled together; comparing the retention time of a standard linalool. filtered and dried under reduced pressure by rotary vacuum evaporator to yield a dark semisolid residue TLC Bio-autography assay (76 gm). % yield was found to be (7.6) w/w. The TLC bio-autographic assay was used to detect the extract was stored in the refrigerator and fresh solutions most bioactive constituents present in the extract by of the extracts were prepared for further study. modified reported method11-13. C. tamala methanol extract (10 mg/ml) was chromatographed over TLC Phytochemical Screening plates (2.5 mm silica gel G, 60F Merck, Darmstadt, Phytochemical screening of the methanol extract of 254 Germany). The plates were then developed with the leaves was performed to know the presence of the toluene-ethyl acetate 80:20 (v/v) as mobile phase14. active chemical constituents such as sterol, The dried TLC plates were subjected to AChE/ BChE carbohydrate, alkaloids, phenols, flavonoids, 8 enzyme inhibitory activities by spraying with terpenoids, saponins and amino acid . Ellman's reagent. Again, the plate was saturated by Preparation of cinnamon oil spraying DTNB/ATCI reagent for AChE inhibition Dried powdered leaves of C tamala were (1 mM DTNB and 1 mM ATCI); in case of BChE, hydro-distilled in a Clevenger apparatus to obtain instead of ATCI, 1 mM BTCI was used. The plates cinnamon oil. Briefly, leaves powder (100 gm) was were allowed to dry for 5 min. Enzyme solution of soaked in water for 2 hrs and the oil was extracted 3 U/ml was sprayed on it and waited for 5 min to through hydro distillation technique9. The distilled appear a white spots within yellow background. This fluid was collected and the oil was separated from the result was documented immediately for inhibition of upper layer. The oil was then dried over anhydrous the compounds. White spots appeared on the TLC Na2SO4 and stored at 4°C. It was transparent, golden confirmed the true inhibitory activity. Another TLC yellow coloured and with strong smell of spice. The plate was developed in the same way without the yield of the oil was found to be (3.2%) v/w. sample to determine the false-positive reaction15,16 and C. tamala compared with true inhibition for further confirmation. HPLC analysis of cinnamon oil of Cinnamon oil and linalool standard were prepared 96 well microtiter plate method in methanol. The prepared sample was filtered AChE and BChE inhibitory activity of the extract, through a whatman (Germany) NYL 0.45 µm pore cinnamon oil, linalool and standard galantamine was size membrane filter. Further, Chromatographic monitored spectrophotometerically in Bio Rad analysis was performed in reverse phase high 96-well microplate reader (680 XR, USA)17. Substrates DALAI et al.: ANTICHOLINESTERASE ACTIVITY OF CINNAMOMUM TAMALA 693 of ATCI/BTCI, reaction mixture of DTNB, bovine samples. The HPLC chromatogram of standard erythrocyte AChE and equine serum BChE were used as linalool and cinnamon oil has been shown in source of enzyme to measure the cholinesterase activity. (Figs. 1a & b). Thus, the result indicates presence of The reaction was initiated with hydrolysis of the linalool as a biomarker compound in cinnamon oil. substrate ATCI/BTCI to form a resultant product So, the present method could be used to quantify the thiocholine which reacts with Ellman's reagent (DTNB) linalool in cinnamon oil. to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which was yellow in color, TLC bio-autography assay catalyzed by AChE/BChE at a wavelength of 405 nm. Standardized methanol extracts of C. tamala 96-wells plates contained 125µl of 3mM DTNB, 25µl of (10 mg/ml) was spotted in TLC plates. Inhibiting spots 15mM ATCI (BTCI when measured the BChE of C. tamala extract on the TLC plate was developed inhibition), 50 µl of buffer and 25µl of sample was with mobile phase of toluene-ethyl acetate 80:20 (v/v). dissolved in phosphate buffer and the absorbance was Cholinesterase (ChE) inhibitory activity was read at 405 nm every 13 s for 65 s.