Cinnamomum Tamala (Buch.-Ham.) T.Nees & Eberm

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. After, 25 µl of determined by spraying with substrate solution of 0.22 U/ml of AChE or BChE was added in each well ATCI/BTCI, reactive agent DTNB and AChE/BChE and the absorbance was measured at every 13 s for enzyme concentration. After spraying, a white spot 104 s. The experiments were done in triplicate. The % with yellow background was visualized in TLC plate. inhibition of the enzyme activity was calculated by The most prominent spot was observed at the Rf of comparing the rates of reaction of samples relative to 0.68, which is the Rf value of C. tamala methanol blank sample (ethanol in phosphate buffer, pH = 8). extract, and it was not the corresponding false positive Inhibition curves were obtained for each compound by spot. From this it may be assumed that bay leaves have plotting between absorbance and time. The linear potential cholinesterase (ChE) inhibitory activity. regression parameters were determined for each curve 96 well microtiter plate method and the IC values were extrapolated. 50 The enzyme inhibitory activity was measured in a

Statistical analysis 96-well micro plate using a micro plate reader (Benchmark Micro plate Reader, Bio-Rad) at 405 nm Data were expressed as Mean ± SEM. IC50 value was calculated from sigmoidal dose response curve with acetylthiocholine (1 mM) as substrate and (variable slope) obtained by plotting the percentage of inhibition versus the concentrations. All data analaysis were performed by using Graph Pad Prism (4.0) and Excel data sheet (Microsoft office 2007).

Results The preliminary phytochemical screening of the C. tamala methanol extract of leaves showed the presence of sterol, alkaloid, phenolic compound, flavonoid, tannin, terpene and saponin.

HPLC analysis of cinnamon oil of C. tamala HPLC profile of standard linalool and cinnamon oil was developed in RP-HPLC. Retention time (Rt) of linalool about 5.3 min and the correlation coefficient of 0.99 showed a good linearity in concentrations ranging from 0.5 to 25 µg/ml. The percentage of linalool present in cinnamon oil was calculated from the experimental peak areas by interpolation to standard calibration curves. The maximum concentration of linalool content was found to be 0.0328% in cinnamon oil. 210 nm detection Fig. 1-HPLC chromatogram of C. tamala (a) Standard linalool (b) wavelengths were used to quantify linalool in the Cinnamon oil 694 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 4, OCTOBER 2014

Ellman's reagent as DTNB. The IC50 values of cinnamon oil, and its extracts showed significant methanol extract, cinnamon oil, linalool and increment in the enzyme inhibition compared to the galantamine (standard) has been shown in Table 1. standard compound linalool. Cinnamon oil obtained from the C. tamala leaf During phytochemical screening, it was observed showed better AChE/BChE activity than methanol that terpene is one of the phytoconstituents of the extract. Linalool was found to be major biomarker of methanol extract. Lopez and Pascual-Villalobos have the cinnamon oil which showed strong inhibition of AChE with (IC : 55.10 ± 0.191µg/ml). The methanol Table 1- IC50 values of the C. tamala extract, cinnamon oil, 50 Linalool and galantamine against acetylcholinesterase and extract and its cinnamon oil showed AChE inhibitory butyrylcholinesterase (values are expressed as Mean ± SEM, N = 6). activity with IC50 value (108.43 ± 0.331 µg/ml) and (94.54 ± 0.774 µg/ml). In general, methanol extract, AChE inhibition BChE inhibition cinnamon oil, linalool and were found to be more Sample IC50 value (µg/ml) IC50 value (µg/ml) potent on AChE than BChE. Cinnamon oil Methanol extract 108.43 ± 0.331 144.97 ± 1.021 (IC50: 135.56 ± 0.912 µg/ml) and biomarker linalool Cinnamon oil 94.54 ± 0.774 135.56 ± 0.912 (IC50: 92.46 ± 0.487 µg/ml) were found to be the most Linalool 55.10 ± 0.191 92.46 ± 0.487 potent inhibitors of BChE. Methanol extract Galantamine 29.15 ± 0.075 34.78 ± 0.364

(IC50: 144.97 ± 1.021 µg/ml) was least active on Values are Mean ± SEM, N= 6 (N = Number of replicates) BChE. Reference compound galantamine were found to be most potent inhibitor on BChE (IC50: 34.78 ± 0.364 µg/ml) then AChE (IC50: 29.15 ± 0.075 µg/ml). In order to further characterize the inhibition potential, the dose response relationships of the acetylcholinesterase and butyrylcholinesterase inhibition have been shown in (Figs. 2 & 3). A dose dependent AChE/BChE inhibition enzyme was observed with the extract, cinnamon oil, linalool and standard compound used. The linear plot showed that linalool has higher % inhibition value comparing to the standard galantamie (Figs. 2 & 3).

Discussion AChE/BChE are the chief enzymes which involves in symptom generation and underlying progression of Fig. 2-Dose response curve of C. tamala methanol extract, 18, 19 cinnamon oil, linalool and galantamine against AD . In progression of AD, concentration of acetylcholinesterase AChE is lost up in specific brain regions; whereas BChE levels chiefly the G1 form, rise with in hydrolyzing excess of Acetylcholine (ACh)20. Though, the specificity of BChE is very less towards hydrolyzing ACh but it is generally viewed as an analogue of AChE, thus it plays an important role in aggravating AD, especially at a very low concentration of AChE21. BChE is an enzyme which catalyses the hydrolysis of ACh, a cholinergic neurotransmission regulates in AD progression. Thus, the BChE inhibition emerged to be another approach to intervene in the progression of AD. Literature study reveals that AChE/BChE inhibitors of plant origin are well utilized for the management of mild to moderate form of AD21. In the present study BChE inhibitory Fig. 3-Dose response curve of C. tamala methanol extract, activity was targeted along with the AChE inhibitory cinnamon oil, linalool and galantamine against activity of bay leaf. % inhibition activity of the butyrylcholinesterase DALAI et al.: ANTICHOLINESTERASE ACTIVITY OF CINNAMOMUM TAMALA 695

been reported that linalool, monoterpenoid chewed on mouth to keep away bad odour. compound showed a reversible competitive AChE Traditionally, in Punjab, leaves of the extract have inhibition22. Linalool is a terpene alcohol compound been used in treament of rheumatism, colic pain and it is widely used in purification process of and diarrhoea29. flavour and fragrance obtained from natural The plant parts have been used traditionally for products23. Linalool is an important phytomarker of its different activities i.e carminative, anthelmintic, bay leaves, as evidenced from the HPLC diuretic, colic, dyspepsia, and diarrhoea, which standardization and ChE inhibitory activity. These have also been verified scientifically as well. findings suggested that AChE/BChE inhibitory Eswaran et al. (2010) studied the gastroprotective activity of the methanol extract and its cinnamon oil activity of an hydro-alcoholic (ethanol 50%) extract may be due to the monoterpenoid constituents. of C. tamala leaves in ethanol (EtOH), The results of the present study also indicated that cold-restraint stress and pylorus ligation induced cinnamon oil of bay leaves was found to be more ulcers in rats at successive dose label of 50,100 and active in comparison methanol extract. This may be 200 mg/kg, p.o, twice daily for 5 days. Pretreatment due the maximum content of linalool in the with C. tamala extract significantly decrease gastric cinnamon oil of C. tamala. Further finding suggest ulcer in dose dependent manner and simultaneously that the affinity of bay leaves was more towards the shows antioxidant activity. The experimental study AChE than BChE. Plants therefore provide useful shows that the gastropotective effect of C. tamala resource of therapeutic lead compounds24,25. Bay leave extract in rats due to decrease level of leaves and its oil may be explored further as a H+K+ATPase activity, volume of gastric juice, acid potential lead in development of anticholinesterase output of the affected region and increase the level inhibiting agents for the management of Alzheimer's of catalytic enzyme in mucus membrane of gastric disease. wall30.

Traditional significance of study to the farmers/society/ The leaves of the C. tamala has been used in Indian researchers and some constructive recommendations ayurvedic medicine as nerve tonic to brain and also an Leaves of C. tamala are widely employed as ingredient of cyavana’s elixir, a rejuvenating liquid spice. The essential oil obtained from it after herbal preparation used for repairing of nervous tissue distillation are very useful in traditional medicne and associated disorder31. A wide range of bioactive and used for several ailments especilaly by people components from leaves and essential oil of C. tamala in Northen part of India. The plant species is also extract have been isolated and most of them have considered as a potential source of economic value shown useful biological activities belonging mainly to farmers by supplying the raw material to local polyphenolic, monoterpenes, sesquiterpene and industries. The dried leaves of the plant has been flavonoid. Recent examination of several extensively used in traditional practice by Manipuri monoterpenoids compounds have revealed the potent (India) farmer to protect the food crops against pest acetylcholinesterase activity against pest insect, manifestation. This common practice has been among them fenchone, γ-terpinene, geraniol and observed in the farmers of Manipur, India. This is linalool have shown highest acetylcholinesterase due to the fact that the leaves of the plant produces inhbition activity22. Our result also suggest that an odour that repelled the agriculture insects in linalool, monoterpenoid compound, a prominent paddy crops26. Some traditonal healers and rural bioactive constituents of essential oils and extract of farmers of many parts of India, also advise to take C. tamala leave have shown strong cholinesterase the mixture of three to four pieces of Tejpata (C. inhbition activity by in vitro study. Cholinesterase tamala), 50 gm Ada (Zingiber officinale) and (acetylcholinesterase and butyrylcholinesterase) Aswatha (Ficus religiosa) leave extract along with inhibitors are the current clinical strategy and USA water to reduce cough and cold in domestic FDA approved compounds used for treatment of animlas27. Over the years, traditional healer of Alzheimer’s disease7. Several plant species have been Uttarakhand region (India) are prescribing mixtue proved to be effective in CNS and related disorder by of water and leaf powder (5 gm) of this plant, taken variety of in vitro and in vivo study, and their on daily basis to cure diabeties condition28. Due to mechanisms of actions have been well established its aromatic character, it is kept in clothes and also through neurochemical approaches. 696 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 13, NO. 4, OCTOBER 2014

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