Solvent preextraction influenced to coumarin and glucose binding capacity of cinnamomi's extracts : Journal of Advanced Pharmaceutical Technology & Research

Secondary Logo

Journal Logo

Original Article

Solvent preextraction influenced to coumarin and glucose binding capacity of cinnamomi's extracts

Ervina, Martha1,2,; Wea, Virgiana Kristin Coco2; Oktaviani, Annisah2; Hartanti, Lanny2; Sinansari, Restry2; Wilianto, Yufita Ratnasari3; Steven, 4; Caroline, 2

Author Information
Journal of Advanced Pharmaceutical Technology & Research 14(2):p 69-74, Apr–Jun 2023. | DOI: 10.4103/japtr.japtr_671_22
  • Open



Cinnamomum burmanii is widely used in Asia not only as flavoring, but also as medicines to antioxidant, anti-inflammatory, and antimicrobes.[1] Moreover, it has been noted in the preclinical to clinical stages of its used for type 2 diabetes and obesity. The cinnamon's water extracts major compounds were cinnamaldehyde, coumarin, and proanthocyanidin type A.[2] The cinnamaldehyde and coumarin are related compounds to its flavor, although the latter has detrimental effects since of its hepatoxic, gene toxic, to carcinogenic, and mutagenic in the animal study. Therefore, the maximum level of coumarin in food and beverages is regulated, especially for Cinnamon containing products. The European Food Safety Authority has established the coumarin's tolerable daily dose of 0.1 mg/kg body weight.[3,4]

For the purpose of its separation, coumarin can be extracted best with 80% methanol. For the purpose of the assay, the spectrophotometry can be used for its fairly selective, sensitive, and inexpensive method.[5,6] The ultraviolet or fluorescent or NaOH spot viewer may be used.[7] The previous study found that Cinnamomi's infusion and ethanol soxhletation provided the best yields and antioxidants capacity compared to ethanol extract with percolation methods as well as to their n-hexane and ethyl acetate fractions.[8] Their antioxidant and antidiabetic potency were also better even compared to rutin and acarbose. Furthermore, noted that the Cinnamon infusion and extracts optimization to their α-glucosidase inhibition.[2]

Therefore, the aim of this study was to continue the process of purifying cinnamon extract and infusion from coumarin by chloroform preextraction to dried plant. It was expected to found a simpler way to produce free or less coumarin extract, thereby reducing unwanted side effects and fulfilled the specified requirements.


Materials and instruments

The dried plant of Cinnamomi (C. burmanii) Cortex was obtained from Tawangmangu (1200 ASL), determined and deposit at the Traditional Medicine Research Center Laboratory (Documented No. Cb-Tw100921), Widya Mandala Catholic University. The chemicals are provided by a local supplier, others cited was used as pro analysis grade. The thin-layer chromatography (TLC) silica gel 60F254, coumarin (E Merck), methanol, chloroform, and ethanol 96% (RDH). The instruments consist of water bath, rotary vaccum evaporator, Multiskan Go microplate reader (Thermo Fisher), and TLC-densitometry (Camag and Switzerland), liquid chromatography–mass spectrometry (LC–MS) (ACQUITY UPLC® BEH).

Material preparation

Cinnamon bark obtained from Tawangmangu, Central Java, Indonesia. Identified and classified as C. burmannii. The bark was cleaned and then standardized, followed by national herbal pharmacopeia.


The Cinnamon bark dried crumb was preextracted three times with chloroform to provide Cinnamon preextracted (P), whereas another named normal Cinnamon (N) for conventional dried Cinnamons. The P and N were then each proceed to infusion following the method by weighing 100 g, then were put into an infusion pot and added with 500 ml of distilled water, heated at 98°C for 20 min, filtered and evaporated using a rotary vacuum evaporator, and water bath to obtained dried extract (PI and NI).[2] While for soxhletation was used 100 g of Cinnamon's bark was objected to ×2.5 circulation, which approximately 250 mL ethanol to produced preextracted (PS) and non preextracted (NS) ethanol extract. The water content of dried extracts was done by the gravimetric method.

The validation of coumarin analysis thin-layer chromatography-densitometry

The validation method followed USP 43, which includes selectivity, linearity, accuracy and precision, limit of detection (LOD), and limit of quantification (LOQ).[9]


A coumarin standard, standard and matrix solutions were made on 3000 ppm and spotted on the TLC plate. The plate was eluted with three systems which were n-hexane: ethyl acetate (8:2), chloroform: methanol (8:2), and ethyl acetate: methanol: water (20:3:2). The spots were observed in their spectrum, wavelength, Rf, and tailing factor (Tf).


A coumarin solution was made on 1500–4500 ppm and spotted on a TLC plate. The experiments were carried out for three times replication, in 3 consecutive days with a selected mobile phase. The regression equation and % coefficient of variation (CV) results were calculated in intraday and interday. The relationship can be declared linear if r count > r table.

Accuracy and precision

The solutions were made 2400, 3000, and 3600 ppm, then added 100 mg of the sample matrix and dissolved in 10 ml of solvents. The solutions were spotted on a TLC plate and eluted. Then, calculate % recovery for accuracy and % CV or relative standard deviation (RSD) for precision.

Limit of detection and limit of quantification

A standard solution was made on 500–1000 ppm, then spotted on a TLC plate and eluted with the selected mobile phase. The area of the selected wavelength is observed and the regression equation is calculated.

Determination of coumarin levels by thin-layer chromatography-densitometry

The infusion and extract were each made on 10,000 ppm and 10 μL of the solution was spotted and eluted. The coumarin's spots are determined and counted for coumarin content.

Liquid chromatography–mass spectrometry/mass spectrometry

The extracts were then subjected (1 μL) to LC–MS analysis with cinnamaldehyde and coumarin standards to the profile of their contents. The system was use waters Acquity UPLC I-Class dan XEVO G2-XS QTof ACQUITY UPLC® BEH C18 1.7 μm 2.1 mm × 50 mm, m/z 100–1200 mode ESI, Solvent A: H2O + 0,1% formic acid (FA) Solvent B: ACN + 0,1% FA.

Glucose-binding capacity

The glucose absorption capacity was measured with slight modification to the Trinder reaction of glucose oxidase-peroxidase aminoantipyrine (Glory Diagnostic, Spain). The samples of extracts were prepared with dimethyl sulfoxide (DMSO) at 5000 ppm, whereas the reference of acarbose and rutin was at 1000 ppm. The blank of each sample was made to correct their origin absorption. To the sample was added glucose 1000 ppm. The glucose-binding capacity was calculated by remain glucose in the samples with spectrophotometer at λ500 nm. The data were presented by mean and standard deviation values of triplicate experiments.


Cinnamon bark is widely used as raw material for the food and beverages, cosmetics to medicinal purposes. Well-known Cinnamon's cinnamaldehyde and coumarin contents are two major secondary metabolites related to its uses. Although latter should be controlled since of its negative effects to some organs. This study was conducted to determine the preextraction influenced to coumarin content in the water infusion and ethanol extract. The cinnamon bark used was observed macroscopically and identifies as C. burmannii. It has rolled shape and brown color with specific odor. It was ground into crumbs and subjected to extractor with water infusion and ethanol soxhletation; as a single to continuous extraction process comparison. The results then evaporate to produce free solvent extracts. Their yield is listed in [Table 1]. The continued extraction by soxhletation was produced a higher yield (19.63 ± 0.26 and 19.24 ± 0.12%) than the water infusion (12.14 ± 0.06 and 11.89 ± 0.05%). The soxhletation was known as exhaustive extraction; with repetitive cycles solvents from the siphon. On the contrary, the preextraction was produced slightly lower than normal infusion and soxhletation. This might cause by some compounds was extracted to chloroform.

Table 1:
The yield and coumarin content results of the extracts

The TLC-densitometry was rapid and less expensive yet suitable for simultaneous coumarin analysis compared to other methods. The validation method for coumarin TLC-densitometry analysis was examined based on the USP 43. The validation method resulted as written in [Table 2]. The selectivity test aimed to determine the ability of a method to measure certain substances carefully and thoroughly in the presence of other components in the sample matrix.[10] Among the three systems, the n-hexane: ethyl acetate gave the best separation of purple spot, with no tailing, and at λmax 285 nm Rf 0.33 ± 0.01. Meanwhile, others resulted in Rf >0.8. This showed that the prior system was optimum, which met the requirements for a Rf value of 0< Rf <1 and the Tf 1 value.[11]

Table 2:
Results of coumarin's validation method

The linearity test is carried out to ensure a linear relationship between analyte concentration and absorbance in such replications and days manners. This was done to obtain the random error and the nonuniformity of the analysis results from the test with the same conditions and at different times. The linearity test is shown in [Table 2] in a range % CV intraday an interday of 0.2653–1.5936, consecutively. A linear relationship between intraday and interday experiments was obtained. The calibration curve for interdays did not give a different value R2 was 0.99541 (y = 4209.345 + 15.6712x) and 2% CV.[12] Based on these and the T statistical test showed not a significant difference.

The accuracy and precision tests are carried out to definiteness and exactness of the result values. The accuracy was expressed as percentage recovery of added analytes; thus determine the effect of the matrix (additive) on the analyte concentration. The precision was expressed as standard deviation or RSD (CV) for each calibration level.[12] The results of the % recovery and % CV tests for infusion and ethanolic extract met the requirements as presented in [Table 2]. The SD and % CV were in the range of 0.32–1.30. These low values presented the method is precise and accurate.

The LOD and LOQ were carried out to determine the presence of the lowest coumarin content in the sample that can be detected and quantified accurately and thoroughly. The results can be seen in [Table 2]. This indicates that the Cinnamon's coumarin concentration in the sample can be observed versus signal noises.[13] The extract's spectra purity was determined by scanned the spots at Rf 0.03 ± 0.01 to reference and by overlaying them at peak's initial, apex, and end positions. [Figure 1 and Table 2] present the overlapped of coumarin's spectra in the extracts and its validation results.

Figure 1:
The overlay spectrum of coumarin's spots on Rf 0.33 in infusion and extract

The coumarin content in preextraction water extract cannot be detected; while counted in the ethanolic soxhletation. This suggested chloroform elimination coumarin; while in soxhletation method showed no significant difference between preextraction and non preextraction. Regarding the result, the water infusion was more preferable than ethanol soxhletation. The water has polar characteristics so that the coumarin's spots in the infusion were hardly observed. As has been known that coumarin is very soluble in ether, diethyl ether, chloroform, oil, soluble in ethanol and methanol, and slightly soluble in water.

Moreover, the study compared the samples capacity to bind glucose at a lower concentration (5.55 mM) to rutin and acarbose [Figure 2]. The glucose-binding activity was showed that reference rutin [R] and acarbose [A] [Figure 3c and d] resulted in higher than all extracts as much as 33%–36% compare to 17%–31%. Thus the preextraction solvents eliminated some content which beneficial effect to bind glucose. Thereby have potential capacity in reducing glucose intestinal absorption in blood glucose. The previous finding obtained the Caesalpinia bonducella (Caesalpiniaceae) had the highest capacity of 74% at 100 mM. The viscosity of soluble polysaccharides or phytochemical content from the extracts may influenced the delay of glucose absorption in the gastrointestinal tract.[14,15]

Figure 2:
The glucose-binding activity of Cinnamomi's extracts and reference
Figure 3:
The structure of Cinnamomum burmannii contents and reference compounds

The LC–MS analysis aimed to examine the extract's profile further; regarding their coumarin and cinnamaldehyde contents as seen in [Table 3 and Figure 4]. The LC–MS profile of the extracts obtained coumarin content in all extracts at varied levels; meanwhile, the cinnamaldehyde was only observed in ethanol (PS and NS) extracts. The LC–MS analysis obtained different results regarding coumarin content compare to TLC-densitometry analysis. The LC–MS has better sensitivity than TLC-densitometry. The PI and NI profiles were similar to reported by Ervina et al.[2] that highlighted the C. burmanii's peaks. They have proposed compounds such as coumarins and A-type proanthocyanidins that major content in and characterized of C. burmanii compare to others species.[16] There were observed similar patterns of PI and NI; PS and NS. The coumarin at Rt 4.33 m/z 147.0440 was observed in all extracts. Importantly noted that the preextraction reduced coumarin 20%–50% to both methods (NI to PI 269092; 132740; NS and PS 545072; 463713, respectively). The cinnamaldehyde volatile oil was observed in PS and NS but not in PI and NI. This phenomenon was also observed to previous results.[2] The catechin-(4α→8)-catechin with m/z 579.1498 was detected on PI and NI but not in PS and NS. The compounds at m/z 865 were recorded on all extracts, which suggested the type A proanthocyanidin.[2] The type A proanthocyanidin and catechin were shared a similar structure base as polyphenol flavan-3-ol [Figure 3a and b], which has been reviewed in vitro and in vivo to clinically used for the prevention and management on type 2 diabetic. The compound possibly has a molecular mechanism by promoting functions and viability of pancreatic β-cells, and insulin signaling pathway for improving glucose transport in muscle and adipose tissue, enhancing the incretin effect, and decreasing of endogenous glucose production.[17]

Table 3:
The LC-MS Summary results
Figure 4:
The LC results of the PI, NI infusions, PS, NS Soxhlet extraction, coumarin, and cinnamaldehyde (a) PI, (b) NI, (c) SP, (d) NP, (e) coumarin, and (f) cinnamaldehyde


The research found that (1) The TLC-densitometry method has met the validation requirement for coumarin analysis that resulted in purple spot at λmax 285 nm, Rf 0.33; obtained intra- and inter-day linear relationship and %CV in a range of 0.2653–1.5936; LOD and LOQ on 52.74 and 175.81 μg/ml, respectively, (2) the chloroform preextraction may reduce coumarin 20%–50% content in the water and ethanolic of Cinnamomi Cortex extracts, and reduced their glucose binding capacity. These results may use further to produce Cinnamomi's free coumarin extract.

Financial support and sponsorship

The Ministry of Education, Culture, Research, and Technology and Widya Mandala Catholic University Surabaya.

Conflicts of interest

There are no conflicts of interest.


Advanced Characterization Laboratories Serpong, National Research and Innovation Institute through E-Layanan Sains, Badan Riset dan Inovasi Nasional for the facilities, and technical support.


1. Singletary K. Cinnamon update of potential health benefits Nutr Today. 2019;54:42–52
2. Ervina M, Lie HS, Diva J, Caroline, Tewfik S, Tewfik I. Optimization of water extract of Cinnamomum burmannii bark to ascertain its in vitro antidiabetic and antioxidant activities Biocatalysis and agricultural Biotechnology. 2019;19:101152
3. European Food Safety Authority (EFSA). . Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the European commission on coumarin in flavourings and other food ingredients with flavouring properties EFSA. 2008;793:1–15 []
4. BfR (Federal Institute for Risk Assessment). . Consumers, Who Eat a Lot of Cinnamon, Currently Have an Overly High Exposure to Coumarin; BfR Health Assessment No. 043/2006 2006 Berlin, Germany BfR Available from: [Last acessed on 2022 Mar 18].
5. Sproll C, Ruge W, Andlauer C, Godelmann R, Lachenmeier DW. HPLC analysis and safety assessment of coumarin in foods Food Chem. 2008;109:462–9
6. Lončar M, Jakovljević M, Šubarić D, Pavlić M, Buzjak Služek V, Cindrić I, et al Coumarins in food and methods of their determination Foods. 2020;9:645
7. Vianna D, Corvello F, Rodio C, Bruxel F, Velho A, Carvalho ES, et al Spectrophotometric Determination of Coumarins Incorporated into Nanoemulsions Containing Pterocaulon balansae Extract Lat Am J Pharm. 2011;30:1487–91
8. Ervina M, Nawu YE, Esar SY. Comparison of in vitro antioxidant activity of infusion, extract and fractions of Indonesian Cinnamon (Cinnamomum burmannii) bark Int Food Res J. 2016;23:1346–50
9. . United States Pharmacopeia 202043th Twinbrook Parkway, Rockville The United States Pharmacopeial Convention
10. Rao TNStaufer M. Validation of analytical methods Calibration and Validation of Analytical Methods – A Sampling of Current Approaches. 2018 United States of America University of Pittsburgh at Greensburg:131–41
11. Reich E, Schibli A. High Performance Thin-Layer Chromatography for the Analysis of Medicinal Plants 2007 Stuttgart Georg Thieme Verlag KG
12. Korhale RV, Chaudhari PH, Wagh N, Maurya P, Singh R, Pimple S, et al Analytical assay method validation and forced degradation behaviour of S(-) amlodipine besilate and losartan potassium from fixed dose combination Int J Univers Pharm Bio Sci. 2014;3:280–95
13. Bhagyasree T, Neelam I, Ajitha A, Rao UM. A review on analytical method development and validation Int J Pharm Res Anal. 2014;4:444–8
14. Bhutkar M, Bhinge S, Randive D, Wadkar G, Todkar S. Studies on glucose adsorption capacity of some indigenous plants Glob J Pharm Sci. 2018;5:555651 10.19080/GJPPS.2018.05.555651
15. Chau CF, Chen CH, Lin CY. Insoluble fiber-rich fractions derived from Averrhoa carambola: Hypoglycemic effects determined by in vitro methods Lebensm Wiss Technol. 2004;37:331–5
16. Chen P, Sun J, Ford P. Differentiation of the four major species of cinnamons (C. burmannii, C. verum, C. cassia, and C. loureiroi) using a flow injection mass spectrometric (FIMS) fingerprinting method J Agric Food Chem. 2014;62:2516–21
17. Márquez Campos E, Jakobs L, Simon MC. Antidiabetic effects of flavan-3-ols and their microbial metabolites Nutrients. 2020;12:1592

Cinnamomum; coumarin; preextraction; thin-layer chromatography-densitometry; validatio

© 2023 Journal of Advanced Pharmaceutical Technology & Research | Published by Wolters Kluwer – Medknow