Nepal J Biotechnol. 2 0 2 1 D e c . 9 (2):14-20 Research article DOI: https://www.doi.org/10.54796/njb.v9i2.41909 ©NJB, BSN 14 Phytochemical Analysis and α-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala Rasna Maharjan1 , Prakash Thapa1 , Karan Khadayat 1, 2 , Surya Kant Kalauni1 1Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal 2Department of Biotechnology, National College, Tribhuvan University, Naya Bazar, Kathmandu, Nepal Received: 5th Nov 2020; Revised: 22nd Sep 2021; Accepted: 6th Dec 2021; Published online: 31st Dec 2021 Abstract The bioactive chemical components of the plant's origin have been used as primary remedies for a wide array of human diseases including diabetes. The present research deal to evaluate and compare anti-diabetic potential of ethanolic and methanolic, young and mature leaves of medicinally valuable Cinnamomum tamala. Total phenolic and flavonoid contents of young and mature leaves were determined. In vitro α-amylase inhibition was carried out using 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) as substrate. Phytochemical screening revealed the presence of polyphenols, flavonoids, terpenoids, quinones, carbohydrates, glycosides, diterpenes, tannins, and reducing sugars. The highest total phenolic content and flavonoid content were observed in methanolic extract of mature leaves (13.725 ± 0.54 mg GAE/g) and young leaves (12.591 ± 0.71 mg QE/g) respectively. Methanolic young leaves extract showed α-amylase inhibition with IC50 value 224.6 ± 2.76 μg/mL as compared to acarbose with IC50 value 5.93 ± 0.14 μg/mL. The result suggests that young leaves of C. tamala had anti-diabetic activity so further work should be carried out. Keywords: α-Amylase, Anti-diabetic, Cinnamomum tamala, Phytochemicals Corresponding author, email: karankhadayat55@gmail.com; skkalauni@gmail.com Introduction Diabetes mellitus is one of the most common health issues among the serious health concerns around the world. It is a chronic multifactorial endocrine disorder of glucose intolerance. Approximately 463 million adults (20-79 years) suffered from diabetes worldwide in 2019 and may escalate to 700 million by 2045. In Nepal, about 696,900 adults are living with diabetes [1]. Among three type of diabetes, Type 1 diabetes occur due to insulin deficiency, type 2 diabetes occur due to inability to utilize insulin and gestational diabetes occur during pregnancy condition [2]. Among them, diabetes of type 2 is the most severe and fast-growing in most countries majorly due to rapid urbanization, diet, lifestyles [3,4]. In hyperglycemia, the rise in blood glucose levels generates excessive superoxide anions which produce hydroxyl radicals through Haber Weiss reaction that can lead to the peroxidation of membrane lipids, oxidative protein damage to cell membranes, and also affects other biomolecules including carbohydrates, proteins, and DNA [5]. Consequently, long-term diabetes leads to stroke, blindness, heart attack, kidney failure, amputation, and the menace of dying prematurely [2]. In recent years, the therapeutic approach for diabetes patients is diet control, exercise, and hypoglycemic drugs such as sulphonylureas, acarbose, and insulin with undesirable side effects such as liver toxicity, lactic acidosis, and diarrhea [6]. In this regard, inhibition of key carbohydrate digestive enzymes (α-amylase) is considered one of the best therapeutic techniques for the treatment of diabetes and its associated diseases. Pancreatic α-amylase is also known as 1, 4-α-D-glucan glucanohydrolase EC (3.2.1.1) plays a vital role in the digestion of starch molecules in the human body. At the first stage, there is partial digestion of starch by salivary amylase enzyme. The enzyme cleaves polymeric substrate (Starch) into shorter oligomers. At the second stage, these shorter oligomers are further split into maltose, maltotriose, and small malto-oligosaccharides with the help of pancreatic α- amylase in the gut [7]. In this present study, α-amylase catalyzes the endohydrolysis of α-1, 4 glycosidic linkages of 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) to yield free chromophore, 2-chloro-nitrophenol (CNP) and release 2-chloro-4-nitrophenyl-α-D-maltoside (CNPG2), maltotriose (G3), and glucose (G). The rate of increase in chromophore absorbance is related to the α-amylase activity of the sample [8,9]. In recent years, the scientist has been so much attracted on the exploration of the secondary metabolites of plants as inhibitors for most effective formulation aspect of drugs with few or hardly negative effects. The medicinal plant contains various natural antioxidants such as tocopherols, vitamin C, and Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 Journal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 https://orcid.org/0000-0003-4460-137X https://orcid.org/0000-0002-3559-9215 https://orcid.org/0000-0002-8125-9756 mailto:karankhadayat55@gmail.com https://orcid.org/0000-0002-4882-4864 mailto:skkalauni@gmail.com Nepal J Biotechnol. 2021 Dec.9 (2):14-20 Maharjan et al. ©NJB, BSN 15 phenolic compounds which are capable to neutralize oxidative damage and protecting from various diseases including diabetes [10]. Plants have been the primary medicinal source for centuries to cure a wide array of human diseases, particularly in developing countries like Nepal due to scarce resources, affordability, and insufficient access to conventional treatment. Thus, considering the extraordinary biodiversity of Nepal, there is a need for scientific investigations of plant species. Among the various medicinal plants, C. tamala is the focus of this research. C. tamala (Buch.-Ham.) T. Nees and Eberm (Luraceace) is one of several traditional remedies used under the Ayurvedic system in Nepal. Its leaves are commonly called tejpat and are used as a common ingredient in cooking. This species is a perennial or evergreen tree up to 7.5 m in height and a girth of about 1.4 cm [11]. Young leaves red-brown, smooth and mature leaves sericeous, glabrescent, and rarely glaucous [12]. Leaves are natural food preservatives for pineapple juice [13]. Besides, tejpat is also a traditional dye-yielding plant [14]. It has been traditionally used as an astringent, stimulant, and carminative [15]. C. tamala leaves exhibited significant biological properties with scientific validation including antioxidant [16,17], antimicrobial [18,19,20], anti-diabetic [21,22,23], anticancer [24], and anti-inflammatory [25]. To the best of the authors’ knowledge, no scientific literature was published regarding the comparison of young and mature leaves extracts of C. tamala to evaluate their bioactivities. The aim of this research was therefore to estimate total phenolic and total flavonoid contents along with the evaluation of in vitro anti-diabetic activity of the extracts of C. tamala by the microplate-based method. Materials and Methods Chemicals and reagent Methanol, ethanol, folin-ciocalteu (FC) reagent were purchased from Qualigens, sodium carbonate, dimethyl sulphoxide (DMSO), potassium acetate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, and sodium chloride were obtained from Fisher Scientific, gallic acid, and quercetin from Hi-media, aluminum chloride from Merck and acarbose, α-amylase from porcine pancreas, and 2- chloro-4-nitrophenyl-α-D-maltotrioside were purchased from Sigma. Collection and authentication of plant materials Fresh young and mature leaves of C. tamala were collected separately from Machhegaun, Kathmandu in July 2019. A herbarium specimen was deposited at the Central Department of Botany, Tribhuvan University, and voucher code RM001 was provided. Extraction The collected young and mature leaves were washed properly with water, shade dried, and ground into powder. The extraction was done by cold percolation method. In short, the powder of young and mature leaves was soaked in methanol and ethanol for 72 hrs with occasional shaking at room temperature. After 72 hrs, the solvent was filtered and the filtrate was evaporated using a rotary evaporator under vacuum at 400 C [26]. The percentage yield was calculated by the given formula: % Yield = Dry weight of extract Dry weight of a plant × 100 Phytochemical screening The presence of phytochemicals in the young and mature leaves of C. tamala was analyzed through the following standard protocol [27, 28, 29]. Estimation of Total Phenolic Content (TPC) Crude extract of young and mature leaves of C. tamala was estimated for TPC by using FC reagent [30,31,32]. In brief, 20 μL sample was added with 100 μL Folin- ciocalteu (2 N), accompanied by 80 μL Na2CO3 (1N). The reaction mixture was incubated in dark at room temperature for 15 minutes until the dark blue color was observed. Finally, the absorbance was taken at 765 nm using a spectrophotometer (Synergy LX, BioTek, Instruments, Inc., USA). A standard calibration curve for gallic acid (10-100 μg/mL) was prepared and phenolic content was expressed as milligrams of gallic acid equivalent per gram of dry weight of the extract (mg GAE/g). All experiments were performed in triplicate and expressed as mean ± standard error of mean. Estimation of Total Flavonoid Content (TFC) Crude extract of young and mature leaves of C. tamala was estimated for TFC by using the aluminum trichloride (AlCl3) method [32,33]. Briefly, 20 μL sample with 110 μL distilled water was added with 60 μL ethanol, 5 μL AlCl3 (10%), and 5 μL potassium acetate (1 M). The reaction mixture was incubated in dark at room temperature for 30 minutes. After incubation, absorbance was measured at 415 nm using a spectrophotometer (Synergy LX, BioTek, Instruments, Inc., USA). A standard calibration curve for quercetin (10 - 100 µg/mL) was prepared and Nepal J Biotechnol. 2021 Dec.9 (2):14-20 Maharjan et al. ©NJB, BSN 16 flavonoid content was expressed as milligrams of quercetin equivalent per gram of dry weight of the extract (mg QE/g). All experiments were done in triplicate and expressed as mean ± standard error of mean. In vitro α-amylase inhibition assay The in vitro α-amylase inhibition was performed in a microplate reader as previously described method [34]. Firstly, young and mature leaves extract was prepared using 50% DMSO, and the reaction was carried out using 50 mM sodium phosphate buffer (pH 7) with 0.9% NaCl. Concisely, 20 µL of extract of different concentrations (31.25 - 1000 µg/mL) was mixed with 80 µL of the enzyme at a final concentration of 1.5 U/mL. Then, it was pre- incubated at 37 °C for 10 min and after pre-incubation, 100 µL CNPG3 substrate at a final concentration of 1 mM was added and left for 15 minutes at the same temperature. The absorbance was measured at 405 nm. DMSO (Not more than 5% was taken as final concentration) was taken as negative control and acarbose as a positive control. All experiments were carried out in triplicate. % Inhibition = A(control) − A(sample) A(control) × 100 A (control) = Absorbance of enzyme-substrate with 50% DMSO A (sample) = Absorbance of enzyme-substrate with extract as inhibitor. Statistical analysis The data were analyzed by using Gen 5 Microplate Data Collection and Analysis Software of microplate reader and then by MS Excel. The IC50 value of crude extract was calculated by using Graph Pad Prism version 8 software and the results were expressed as mean ± Standard error mean (SEM). Results Phytochemical screening The percentage yield was found to be higher in methanol as compared to ethanol and are given in Table 1. The range of percentage yield of mature and young leaves varies from 7.36 to 18.22%. The young leaf had the highest percentage yield (EEY 10.22 % and MEY 18.22 %) and the mature leaf had the lowest percentage yield (EEM 7.36 % and MEM 12.24 %). The different phytochemical tests had shown the presence of polyphenols, flavonoids, tannins, quinones, carbohydrates, glycosides, terpenoids, and reducing sugar whereas basic alkaloids are absent in both extracts of C. tamala (Table 2). Table 1. Percentage yield of different extract of C. tamala S. No. Sample Percentage yield (%) 1 Ethanolic extract of mature leaf (EEM) 7.36 2 Ethanolic extract of young leaf (EEY) 10.22 3 Methanolic extract of mature leaf (MEM) 12.24 4 Methanolic extract of young leaf (MEY) 18.22 Table 2. Preliminary phytochemical screening of different extract of C. tamala S. No. Phytochemicals MEM MEY EEM EEY 1 Polyphenols + + + + 2 Flavonoids + + + + 3 Tannins + + + + 4 Quinones + + + + 5 Carbohydrates + + + + 6 Glycosides + + + + 7 Terpenoids + + + + 8 Diterpenes + + + + 9 Reducing sugars + + + + 10 Basic alkaloids - - - - “+” indicates presence, “-” indicates absence Total Phenolic and Flavonoid Content The TPC value of ethanolic young and mature leaves extract was 9.83 ± 0.30 mg GAE/g and 8.91 ± 0.39 mg GAE/g respectively whereas, the TPC value of methanolic young and mature leaves extract was 11.26 ± 0.17 mg GAE/g and 13.73 ± 0.55 mg GAE/g. The TFC value of ethanolic young and mature leaves extract was 6.82 ± 0.67 mg QE/g and 7.02 ± 0.32 mg QE/g respectively whereas, the TFC value of methanolic young and mature leaves extract was 12.59 ± 0.71 mg QE/g and 11.84 ± 0.66 mg QE/g as shown in Table 3. Table 3. Total phenolic and flavonoid content of different extract of C. tamala S. No. Sample TPC mg GAE/g TFC mg QE/g 1 MEM 13.73 ± 0.55 11.84 ± 0.66 2 MEY 11.26 ± 0.17 12.59 ± 0.71 3 EEM 8.91 ± 0.39 7.02 ± 0.32 4 EEY 9.83 ± 0.30 6.82 ± 0.67 Mean ± SEM (n = 3) In vitro α-amylase inhibition assay Table 4. Screening for α-amylase inhibition of different extract S. No. Sample Concentration % Inhibition 1 MEM 500 µg/mL < 50 2 MEY 500 µg/mL 70.33 ± 0.47 3 EEM 500 µg/mL < 50 4 EEY 500 µg/mL < 50 5 Acarbose 50 µg/mL 96.77± 0.028 Mean ± SEM (n = 3) The percentage inhibition of young, and mature leaves of C. tamala using methnol and ethanol as solvent were screened for 𝛼-amylase inhibitory activity at 500 µg/mL (Table 4). Young leaf had the highest inhibition of 𝛼- Nepal J Biotechnol. 2021 Dec.9 (2):14-20 Maharjan et al. ©NJB, BSN 17 amylase enzyme whereas mature leaf showed the lowest inhibition at the same concentration in our study. Only methanolic extract of young leaves had shown 70.33% inhibition while screening. So, the methanolic extract of young leaves was further assessed for IC50 value at different concentrations (Table 5) and was found to be 224.6 ± 2.76 μg/mL. However, compared to standard drug acarbose, the methanolic extract of young leaves showed moderate 𝛼-amylase inhibitory activity (Table 6) and was found to be 5.93± 0.14 as shown in Table 7. Table 5. α-Amylase inhibition of methanolic extract of young leaves (MEY) at different concentration Concentration Inhibition 1 Inhibition 2 Inhibition 3 Mean ± SEM 500 µg/mL 70.15 69.62 71.23 70.33 ± 0.47 250 µg/mL 54.80 58.59 58.38 57.26 ± 1.23 125 µg/mL 33.37 35.45 31.89 33.57 ± 1.03 62.50 µg/mL 14.89 9.46 10.04 11.46 ± 1.72 31.25 µg/mL 2.83 3.78 6.78 4.46 ± 1.19 Mean ± SEM (n = 3) 0 200 400 600 0 20 40 60 80 MEY Concentration (µg/mL) % I n h ib it io n Inhibition 1 Inhibition 2 Inhibition 3 Table 6. α-Amylase inhibition of acarbose at different concentration Concentration Inhibition 1 Inhibition 2 Inhibition 3 Mean± SEM 50 µg/mL 96.82 96.75 96.72 96.76± 0.02 25 µg/mL 90.08 89.93 89.80 89.94± 0.08 12.5 µg/mL 77.72 77.07 76.88 77.22± 0.25 6.25 µg/mL 55.44 51.92 53.38 53.58± 1.02 3.125 µg/mL 24.27 23.77 33.52 27.19± 3.16 1.563 µg/mL 5.33 6.64 4.01 5.33± 0.75 Mean ± SEM (n = 3) 0 20 40 60 0 50 100 150 Acarbose Concentration (µg/mL) % I n h ib it io n Inhibition 1 Inhibition 2 Inhibition 3 Table 7. IC50 values of acarbose and potent sample (MEY) S. No. Sample IC50 (µg/mL) 1 Acarbose 5.93± 0.14 2 MEY 224.6 ± 2.76 Mean ± SEM (n = 3) Discussion Diabetes has been stated as the primary cause of global morbidity and mortality [35]. Retarding the absorption and digestion of carbohydrates in the intestine through inhibition of carbohydrate digestive enzymes such as α- amylase is one of the therapeutic strategies to minimizing postprandial hyperglycemia [36, 37, 38]. This study reported the potential in vitro amylase inhibitory properties of C. tamala mature and young leaf by identifying its main phytochemical constituents. Extraction of phytochemicals is the preliminary process for recovering and isolating compounds from the pulverization of plant materials [39]. The extraction yield depends upon different parameters such as solvent with varying polarity, pH, temperature, extraction time, and sample composition [40]. In our study methanolic extracts showed (MEM, MEY) higher yield than that of ethanolic extracts (EEY, EEM). The difference might be due to the high polarity of methanol that may cause higher solubility of phenolics, flavonoids, alkaloids, and terpenoids compounds in methanol as compared to other solvents including ethanol [40, 41,42]. The phytochemicals obtained from C. tamala are shown in Table 2 that was compared with the previously reported study and found similar phytochemical profiling [43]. Our findings exhibited that methanolic extracts have the higher TPC and TFC relative to ethanolic extracts as seen in Table 3. Aryal et al. suggested that numerous phenolic compounds are present in plant extracts that are responsible for enzyme inhibition supported by TPC (mg GAE/g) and TFC (mg QE/g) of MEM (13.73 ± 0.55, 11.84 ± 0.66), MEY (11.26 ± 0.17, 12.59 ± 0.71), EEM (8.91 ± 0.39, 7.02 ± 0.32), and EEY (9.83 ± 0.30, 6.82 ± 0.67) respectively [44]. Phenolics and flavonoids compounds such as quercetin, ferulic acid, Nepal J Biotechnol. 2021 Dec.9 (2):14-20 Maharjan et al. ©NJB, BSN 18 anthocyanins, catechin, and resveratrol are the reason behind controlling glycemia via α-glucosidase, α- amylase, and lipid peroxidation inhibition, insulin secretion, and accelerating glucose uptake [45,46]. The difference in TPC and TFC is due to different nature of extracting solvents [47]. Previously, it was revealed that younger leaves are most active in biosynthesis and accumulation of phenolic, flavonoid, and proanthocyanidins than the mature leaves of Lantana camara [48]. Similarly in our study, among young and mature leaf extracts, only methanolic extract of young leaf showed significant inhibition toward the α-amylase enzyme. The ethanolic extract of young leaf failed to show significant inhibition that might be due to its less polar nature causing lower yield of phenolics and flavonoids compounds. Besides that, high flavonoid content of MEY might be responsible for high inhibition as compared to other extracts. Several potential α-amylase and α-glucosidase inhibitors from medicinal plants belongs to flavonoid class as reported by earlier study [49]. Phenolic compounds including flavonoids and tannins have been reported to play a vital role in controlling diabetes [50, 51, 52]. Phenolic compounds mainly flavonoids interact strongly with proteins and could inhibit their enzymatic activities by making complexes and changing conformation [53]. The IC50 value of acarbose as positive control showed around 40 times more potency than MEY as shown in Table 5. This might be due to plant extract contains a jumble of multiple compounds as compared to pure inhibitor compound acarbose [34, 54]. Previous studies reported that polyphenol compounds (proanthocyanidins) derived from different plant sources showed potent amylase inhibitors [55, 56]. A-type procyanidin oligomer of cinnamon led to potent antioxidant properties and acts as an insulin sensitizer [57]. Besides that, cinnamtannin D-1 (CD1) isolated from C. tamala was reported to shield pancreatic β-cells from palmitic acid-induced apoptosis and oral consumption of cinnamaldehyde had hypoglycemic and hypolipidemic activity in STZ-induced diabetic rats respectively [23, 58]. These compounds might be present in the extract of young leaves, so further isolation and molecular characterization are needed. Conclusion The present study evaluated phytochemical constituents as well as α-amylase inhibition of methanolic and ethanolic extracts from young and mature leaves of C. tamala. The in vitro results revealed that methanolic extract and young leaves possess higher antidiabetic activity. Thus, it was suggested that further investigation for isolation and characterization of a bioactive compound and its kinetic study from MEY should be carried out that can act as an anti-diabetic agent. Abbreviations GAE (Gallic Acid Equivalent), QE (Quercetin Equivalent), CNPG3 (2-chloro-4-nitrophenyl-α-D- maltotrioside), MEY (Methanolic Extract of Young leaf), MEM (Methanolic Extract of Mature Leaf), EEY (Ethanolic Extract of Young leaf), EEM (Ethanolic Extract of Mature Leaf), TPC (Total Phenolic Content), TFC (Total Flavonoid Content) Author’s contributions This research was performed in collaboration with all authors. RM and KK have contributed to the plan of the research work. The first draft was written by RM, responsible for data analysis, who conducted technical work (Lab). SKK supervised the research project, PT revised the manuscript. The final manuscript was read and approved by all authors. Competing interests No competing interests. Funding The research was completed by financial support from the University Grant Commission (Sanothimi, Bhaktapur, Nepal). Acknowledgments The authors extend their appreciation to the Central Department of Chemistry, Tribhuvan University for providing an opportunity to conduct this research and Prof. Dr. Suresh Kumar Ghimire of Central Department of Botany, Tribhuvan University for plant identification Ethical approval and consent Not applicable References 1. IDF Diabetes Atlas 9th edition 2019 [Internet]. [cited 2020 Sep 2]. Available from: https://www.google.com/search?client=firefox- b-d&q=IDF+Diabetes+Atlas+9th+edition+2019.+%5B Online%5D.%28URL+https%3A%2F%2Fwww.diabetesatlas.org %2Fen%2F%29.+%28Accessed+27+January+2020%9 2. WHO | Global report on diabetes [Internet]. WHO. World Health Organization; [cited 2020 Sep 2]. Available from: http://www.who.int/diabetes/global-report/en/ 3. Rowley WR, Bezold C. Creating Public Awareness: State 2025 Diabetes Forecasts. Popul Health Manag [Internet]. [cited 2020 Oct 30]. Available from: https://www.liebertpub.com/doi/ full/ 10.1089/pop.2011.0053 4. Sansar N. World Diabetes Day 2018: 4% of Nepal Population Diabetic! [Internet]. Nepali Sansar. 2018 [cited 2020 Sep 2]. Available from: https://www.nepalisansar.com/health/ world- diabetes-day-2018-4-of-nepal-population-diabetic/ Nepal J Biotechnol. 2021 Dec.9 (2):14-20 Maharjan et al. ©NJB, BSN 19 5. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40(4):405-12. https://doi.org/10.2337/diab.40.4.405 6. Tahrani AA, Piya MK, Kennedy A, Barnett AH. Glycaemic control in type 2 diabetes: targets and new therapies. Pharmacol Ther. 2010;125(2):328-61. https://doi.org/10.1016/ j.pharmthera .2009.11.001 7. Kumanan R, Manimaran S, Saleemulla K, Dhanabal SP, Nanjan MJ. Screening of bark of Cinnamomum tamala (Lauraceae) by using α-amylase inhibition assay for anti-diabetic activity. Int J Pharm Biomed Res. 2010;1(2):69-72. 8. Sudha P, Zinjarde SS, Bhargava SY, Kumar AR. Potent α-amylase inhibitory activity of Indian Ayurvedic medicinal plants. BMC Complement Altern Med. 2011;11(1):5. https://doi.org/10.1186/1472-6882-11-5 9. Barni F, Berti A, Rapone C, Lago G. α-Amylase kinetic test in bodily single and mixed stains. J Forensic Sci. 2006;51(6):1389-96. http//doi.org/10.1111/j.1556-4029.2006.00259.x 10. Yang J-H, Lin H-C, Mau J-L. Antioxidant properties of several commercial mushrooms. Food Chem. 2002;77(2):229-35. https://doi.org/10.1016/S0308-8146(01)00342-9 11. Majumder P, Paridhavi M. An ethno-phytochemical and pharmacological review on novel Indian medicinal plants used in herbal formulations. Int J Pharm Pharm Sci. 2013;5(4):74-83. 12. Flora of Nepal - Online Flora [Internet]. [cited 2020 Sep 2]. Available from: http://www.floraofnepal.org /onlineflora? name= rossulariaceae&pubname=FloraOfNepal 13. Kapoor IPS, Singh B, Singh G. Essential oil and oleoresins of Cinnamomum tamala (tejpat) as natural food preservatives for pineapple fruit juice. J Food Process Preserv. 2008;32(5):719-28. https://doi.org/10.1111/j.1745-4549.2008.00200.x 14. Gaur RD. Traditional dye yielding plants of Uttarakhand, India. Indian J. Nat Prod Resour. 2008;7:154-265. 15. Malla SB, Shakya PR. Medicinal plants of Nepal. His Majesty's Government of Nepal, Ministry of Forests, Department of Medicinal Plants; 1970. 16. Prasad KN, Yang B, Dong X, Jiang G, Zhang H, Xie H, et al. Flavonoid contents and antioxidant activities from Cinnamomum species. Innov Food Sci Emerg Technol. 2009;10(4):627-32. https://doi.org/10.1016/j.ifset.2009.05.009 17. Sultana S, Ripa FA, Hamid K. Comparative antioxidant activity study of some commonly used spices in Bangladesh. Pak J Biol Sci. 2010;13(7):340. 18. Parekh J. In vitro screening of antibacterial activity of aqueous and alcoholic extracts of various Indian plant species against selected pathogens from Enterobacteriaceae. Afr J Microbiol Res. 2007;1(6):92-9. https://doi.org/10.5897/AJMR.9000402 19. Jeyasree P, Dasarathan P. Screening of phytochemicals and immuneomodulatory potential of amedicinal plant, Cinnamomum tamala. Int J Pharma Sci Res. 2012;3:1049-52. 20. Thombre RS, Shinde V, Thaiparambil E, Zende S, Mehta S. Antimicrobial activity and mechanism of inhibition of silver nanoparticles against extreme halophilic archaea. Front Microbiol. 2016;7:1424. https://doi.org/10.3389 /fmicb.2016.01424 21. Sun P, Wang T, Chen L, Yu BW, Jia Q, Chen KX, Fan HM, Li YM, Wang HY. Trimer procyanidin oligomers contribute to the protective effects of cinnamon extracts on pancreatic β-cells in vitro. Acta Pharmacol Sin. 2016;37(8):1083-90. https://doi.org/10.1038/aps.2016.29 22. Udupa KN, Tripathi SN, Chandola HM. Effect of Cinnamomum tamala on plasma insulin vis-à-vis blood sugar in patients ofdiabetes mellitus. J Res Ayu Siddha. 1980;1:345-57. 23. Wang T, Sun P, Chen L, Huang Q, Chen K, Jia Q, Li Y, Wang H. Cinnamtannin D-1 protects pancreatic β-cells from palmitic acid- induced apoptosis by attenuating oxidative stress. J Agric Food Chem. 2014 Jun 4;62(22):5038-45. https://doi.org/10.1021/jf500387d 24. Shahwar D, Ullaha S, Khan MA, Ahmad N, Saeed A, Ullah S. Anticancer activity of Cinnamomum tamala leaf constituents towards human ovarian cancer cells. Pak J Pharm Sci. 2015;28(3):969-72. 25. Dumbre RK, Kamble MB, Patil VR. Inhibitory effects by ayurvedic plants on prostate enlargement induced in rats. Pharmacogn Res. 2014;6(2):127-32. https://dx.doi.org/10.4103%2F0974-8490.129031 26. Zhang S, Bi H, Liu C. Extraction of bio-active components from Rhodiola sachalinensis under ultrahigh hydrostatic pressure. Sep Purif Technol. 2007;57(2):277-82. https://doi.org/10.1016/j. seppur.2007.04.022 27. Sofowora A. Medicinal plants and traditional medicine in Africa. Ibadan. Niger Spectr Books Ltd. 1993:191-289. 28. Harborne AJ. Phytochemical methods a guide to modern techniques of plant analysis. Springer science and business media; 1998. 29. Evans WC. Trease and Evans Pharmacognosy. 15th ed. Sanders Co Ltd Singap. 2002. 30. Ainsworth EA, Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc. 2007;2(4):875-77. https://doi.org/10.1038/nprot.2007.102 31. Lu X, Ross CF, Powers JR, Aston DE, Rasco BA. Determination of total phenolic content and antioxidant activity of Garlic (Allium sativum) and Elephant Garlic (Allium ampeloprasum) by attenuated total reflectance–fourier transformed infrared spectroscopy. J Agric Food Chem. 2011;59(10):5215-21. https://doi.org/10.1021/jf201254f 32. Sapkota BK, Khadayat K, Adhikari B, Poudel DK, Niraula P, Budhathoki P, Aryal B, Basnet K, Ghimire M, Marahatha R, Parajuli N. Phytochemical Analysis, Antidiabetic Potential and in-silico Evaluation of Some Medicinal Plants. Pharmacogn Res. 2021;13(3):140-48. https:// doi.org/10.5530/pres.13.3.6 33. Chang C-C, Yang M-H, Wen H-M, Chern J-C. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal. 2002;10(3):178-82. https://doi.org/10.38212/2224-6614.2748 34. Khadayat K, Marasini BP, Gautam H, Ghaju S, Parajuli N. Evaluation of the alpha-amylase inhibitory activity of Nepalese medicinal plants used in the treatment of diabetes mellitus. Clin Phytoscience. 2020;6(1):1-8. https://doi.org/10.1186/s40816-020-00179-8 35. Bouguerra R, Alberti H, Salem LB, Rayana CB, Atti JE, Gaigi S, et al. The global diabetes pandemic: the Tunisian experience. Eur J Clin Nutr. 2007;61(2):160–65. https://doi.org/10.1038 /sj.ejcn.1602478 36. Hogan S, Zhang L, Li J, Sun S, Canning C, Zhou K. Antioxidant rich grape pomace extract suppresses postprandial hyperglycemia in diabetic mice by specifically inhibiting α- glucosidase. Nutr Metab. 2010;7(1):1-9. https://doi.org/10.1186/1743-7075-7-71 37. Hamden K, Keskes H, Belhaj S, Mnafgui K, Allouche N. Inhibitory potential of omega-3 fatty and fenugreek essential oil on key enzymes of carbohydrate-digestion and hypertension in diabetes rats. Lipids Health Dis. 2011;10(1):1-10. https://doi.org/10.1186/1476-511X-10-226 38. Mayur B, Sancheti S, Shruti S, Sung-Yum S. Antioxidant and α- glucosidase inhibitory properties of Carpesium abrotanoides L. J Med Plants Res. 2010;4(15):1547-53. https://doi.org/ 10.5897/JMPR.9000218 39. Stalikas CD. Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci. 2007;30(18):3268-95. https://doi.org/10.1002/jssc.200700261 40. Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, et al. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J Food Drug Anal. 2014;22(3):296-302. https://doi.org/10.1016/j.jfda.2013.11.001 41. Duh P-D, Yeh D-B, Yen G-C. Extraction and identification of an antioxidative component from peanut hulls. J Am Oil Chem Soc. 1992;69(8):814-18. https://doi.org/10.1007/BF02637703 42. Gill S, Panthari P, Kharkwal H. Phytochemical investigation of high altitude medicinal plants Cinnamomum tamala (Buch-ham) Nees and Eberm and Rhododendron arboreum smith. Am J Phytomed Clin Ther. 2015;3:512-28. Nepal J Biotechnol. 2021 Dec.9 (2):14-20 Maharjan et al. ©NJB, BSN 20 43. Apak R, Güçlü K, Demirata B, Özyürek M, Çelik SE, Bektaşoğlu B, et al. Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules. 2007;12(7):1496-1547. https://doi.org/10.3390/12071496 44. Aryal B, Niraula P, Khadayat K, Adhikari B, Khatri Chhetri D, Sapkota BK, Bhattarai BR, Aryal N, Parajuli N. Antidiabetic, Antimicrobial, and Molecular Profiling of Selected Medicinal Plants. Evid-Based Complementary Altern. Med. 2021 May 7;2021. https://doi.org/10.1155/2021/5510099 45. Barone E, Calabrese V, Mancuso C. Ferulic acid and its therapeutic potential as hormetin for age-related diseases. Biogerontology. 2009 Apr;10(2):97-108. https://doi: 10.1007/s10522-008-9160-8 46. Rizvi SI, Mishra N. Anti‐oxidant effect of quercetin on type 2 diabetic erythrocytes. J. Food Biochem. 2009 Jun;33(3):404-15. https://doi: 10.1111/J.1745-4514.2009.00228.x 47. T. Van Ngo, C. J. Scarlett, M. C. Bowyer, P. D. Ngo, and Q. Van Vuong, “Impact of different extraction solvents on bioactive compounds and antioxidant capacity from the root of Salacia chinensis L. J. Food Qual. 2017 Jan 11: 2017. https://doi: 10.1155/2017/9305047 48. Bhakta D, Ganjewala D. Effect of leaf positions on total phenolics, flavonoids and proanthocyanidins content and antioxidant activities in Lantana camara (L). J Sci Res. 2009;1(2):363-69. https://doi.org/10.3329/jsr.v1i2.1873 49. Yang S, Meng Y, Yan J, Wang N, Xue Z, Zhang H, Fan Y. Polysaccharide-enriched fraction from Amillariella mellea fruiting body improves insulin resistance. Molecules. 2019;24(1):46. https://doi.org/10.3390/molecules24010046 50. Marrelli M, Amodeo V, Statti G, Conforti F. Biological properties and bioactive components of Allium cepa L.: Focus on potential benefits in the treatment of obesity and related comorbidities. Molecules. 2019;24(1):119. 51. Keskes H, Mnafgui K, Hamden K, Damak M, El Feki A, Allouche N. In vitro anti-diabetic, anti-obesity and antioxidant proprieties of Juniperus phoenicea L. leaves from Tunisia. Asian Pac J Trop Bio. 2014;4:S649-55. https://doi.org/10.12980 /APJTB.4.201414B114 52. Fruit and vegetable intake and incidence of type 2 diabetes mellitus: systematic review and meta-analysis | The BMJ [Internet]. [cited 2020 Nov 3]. Available from: https://www.bmj.com/content/341/bmj.c4229 53. Pochhi ML. An antioxidant activity of Cinnamonum tamala improves histopathological alterations and biochemical parameters in alloxan-induced diabetic rats. Asian J Med Sci. 2019;10(6):50-6. 54. Lavelli V, Harsha PSS, Laureati M, Pagliarini E. Degradation kinetics of encapsulated grape skin phenolics and micronized grape skins in various water activity environments and criteria to develop wide-ranging and tailor-made food applications. Innov Food Sci Emerg Technol. 2017;39:156-64. https://doi.org/10.1016/j.ifset.2016.12.006 55. Taslimi P, Aslan HE, Demir Y, Oztaskin N, Maraş A, Gulçin İ, et al. Diarylmethanon, bromophenol and diarylmethane compounds: Discovery of potent aldose reductase, α-amylase and α-glycosidase inhibitors as new therapeutic approach in diabetes and functional hyperglycemia. Int J Biol Macromol. 2018;119:857-63. https://doi.org/ 10.1016/j.ijbiomac. 2018 .08.004 56. Anderson RA, Broadhurst CL, Polansky MM, Schmidt WF, Khan A, Flanagan VP, et al. Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity. J Agric Food Chem. 2004;52(1):65-70. https://doi.org/10.1021/jf034916b 57. Babu PS, Prabuseenivasan S, Ignacimuthu S. Cinnamaldehyde-a potential antidiabetic agent. Phytomedicine. 2007;14(1):15-22. https://doi.org/10.1016/ j.phymed.2006.11.005