Indian Journal of Health Sciences and Biomedical Research KLEU

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 13  |  Issue : 2  |  Page : 105--111

Qualitative, quantitative, and antioxidant analysis of phytochemicals present in Cinnamomum zeylanicum species


T Sivapriya1, Sheila John2,  
1 Department of Clinical Nutrition, SDNB Vaishnav College for Women, Chennai, Tamil Nadu, India
2 Department of Home Science, Women's Christian College, Chennai, Tamil Nadu, India

Correspondence Address:
Dr. T Sivapriya
Department of Clinical Nutrition, SDNB Vaishnav College for Women, Chennai, Tamil Nadu
India

Abstract

BACKGROUND: Many species of cinnamon are grown throughout the world. The bark of cinnamon has been used as a traditional medicine from ancient times. Scientific proof regarding the presence of active compounds responsible for its medicinal property in Cinnamomum zeylanicum is much needed. AIM: The present study was carried out to evaluate the phytochemical properties and anti-oxidant properties of C. zeylanicum bark. MATERIALS AND METHODS: After preliminary qualitative screening, the aqueous extract of C. zeylanicum was quantitatively assessed for total phenol, flavonoid, tannin, saponin, and coumarin. The antioxidant property was evaluated by ferric reducing antioxidant power (FRAP) assay. RESULTS AND DISCUSSION: C. zeylanicum species was found to possess different amounts of phytochemicals. Among the five components, total polyphenol content was highest in the extract. The total phenol content was around 436 mg/g, followed by saponin which was found to be around 71.25 mg, the tannin content was 43.80 mg, and the amount of flavonoid was 41.92 mg. The coumarin content was 57.70 mg. Estimation of the antioxidant potential by FRAP method indicated the maximum ferric reducing antioxidant power to be 1.377 at 1000 μg. CONCLUSION: Thus, the investigation proved the presence of several active constituents and the antioxidant potential of C. zeylanicum so that it can be used as regular food for preventing and curing diseases.



How to cite this article:
Sivapriya T, John S. Qualitative, quantitative, and antioxidant analysis of phytochemicals present in Cinnamomum zeylanicum species.Indian J Health Sci Biomed Res 2020;13:105-111


How to cite this URL:
Sivapriya T, John S. Qualitative, quantitative, and antioxidant analysis of phytochemicals present in Cinnamomum zeylanicum species. Indian J Health Sci Biomed Res [serial online] 2020 [cited 2020 Sep 28 ];13:105-111
Available from: http://www.ijournalhs.org/text.asp?2020/13/2/105/287423


Full Text



 Introduction



Cinnamomum zeylanicum is used as a spice as well as a traditional medicine from ancient times. Its use has been widely accepted in Indian traditional Ayurvedic medical systems and Chinese medical systems to treat and prevent diseases. A thorough understanding about the phytochemicals present in C. zeylanicum and their properties is vital for finding novel functional foods and nutraceuticals.

There is an increasing interest both in the industry and in scientific research for spices and aromatic herbs because of their strong antioxidant and antimicrobial properties, which exceed many currently used natural and synthetic antioxidants. These properties are due to the phytochemical substances such as phenols, flavonoids, terpenoids, carotenoids, and phytoestrogens which make them an excellent antioxidant food. Antioxidants have a long history of use in the nutrition/health community and food industry.[1]

Secondary metabolites from plants have important biological and pharmacological activities, such as antioxidative, anti-allergic, antibiotic, hypoglycemic, and anticarcinogenic property. Many disorders in human such as diabetes, atherosclerosis, arthritis, Alzheimer disease, and cancer may be the result of increased concentrations of free radicals in an organism. Reactive oxygen species and nitrogen species, as the most frequent pro-oxidants, either originate from normal metabolism or are induced by ultraviolet (UV) radiation and different pollutants. Harmful effects of disturbed antioxidant–pro-oxidant balance can be largely prevented by the intake of antioxidant substances.[2]

Antioxidants have already been found in plant materials and supplements. Due to their natural origin, the antioxidants obtained from plants are of greater benefit in comparison to synthetic ones. The use of natural antioxidants from plants does not induce side effects, while synthetic antioxidants were found to have a genotoxic effect.[3] The basic aim of the research was to screen C. zeylanicum species for the presence of various phytochemicals and then to quantitatively estimate the total phenolic content, concentrations of flavonoids, tannin saponin, and coumarin in aqueous extracts of the species. The objective also included the determination of the antioxidant activity of cinnamon extracts by FRAP assay.

 Materials and Methods



The study protocol was reviewed and approved by the Independent Institutional Ethics Committee of Women's Christian College, Chennai (Ethical clearance No. WCC/HSC/11EC-2014:02). An experimental study design was carried out for qualitative and quantitative estimation of phytochemicals. Ferric reducing antioxidant potential (FRAP) assay was conducted to prove radical scavenging potential.

Cinnamon powder was prepared from the inner bark of C. zeylanicum. About 1 kg of C. zeylanicum inner barks was procured from a traditional medical shop, dried, and cleaned. C. zeylanicum bark was authorized as original cinnamon by “The Astoria Research and Analyticals,” Chennai. The bark was then pulverized with a blender, sieved, and then stored in an airtight container. Aqueous, methanolic, and ethanolic extracts of the bark were prepared in 0.5 g/ml.[4]

Chemicals and reagents

Most of the chemicals used for phytochemical screening were purchased from Biosar, India. Folin–Ciocalteu's reagent was purchased from Loba Chemie as a 2N reagent. Gallic acid, ascorbic acid, and rutin were purchased from Hi-Media, India. Standard compound coumarin (2H-1-benzopyran-2-one), sodium phosphate ferricyanide, and 10% trichloroacetic acid were purchased from Sigma-Aldrich, India.

Preparation of test samples and procedure

The aqueous, methanolic, and ethanolic extracts were subjected to preliminary phytochemical screening to detect the presence of phytoconstituents by the following methods: Test samples (5.0 g each) were transferred to conical flasks and 50 ml of methanol, ethanol, and distilled water were added as a single solvent in each flask. The mixtures were stored for 24 h at room temperature. Finally, the samples were filtered with Whatmann filter paper No. 1 and used for the qualitative analysis.

Detection of alkaloids

To 5 ml of extract, dilute hydrochloric acid was added and filtered. The filtrate was used to perform the following test for the detection of alkaloids.

Mayer's test: Filtrates were treated with Mayer's reagent consisting of potassium mercuric iodide. Formation of a yellow-colored precipitate confirms the presence of alkaloids.

Dragendorff's test: About 1 ml of the filtrate was treated with Dragendorff's reagent. The appearance of a reddish-brown precipitate indicates the presence of alkaloid.

Detection of glycosides

Legal's test

Test sample extracts (1 ml each) were added to individual test tubes and 1 ml pyridine and 1 ml sodium nitroprusside were added to each test tube. The development of pink to red color confirms the presence of glycosides.

Detection of saponin

Froth Test

Extracts were diluted with distilled water to 20 ml and vigorously shaken in a conical flask for 15 min. The formation of foam indicates the presence of saponin.

Foam Test

0.5 g of the extract was shaken with 2 ml of water. If the produced foam continues for 10 min, it indicated the presence of saponin.

Detection of phenols

Ferric Chloride Test

Extracts were treated with 3–4 drops of 1% ferric chloride solution. The formation of bluish-black color confirms the presence of phenols.

Detection of flavonoids

Lead Acetate test

To the test sample, 12–15 drops of lead acetate solution were added. Formation of yellow color precipitate confirms the presence of flavonoids.

Shinoda's test

The bark extracts were dissolved in alcohol. To this, one piece of magnesium and concentrated hydrochloric acid were added dropwise and heated. The appearance of magenta color shows the presence of flavonoids.

Detection of steroids

Liebermann–Burchard test

Samples were treated with concentrated sulfuric acid, and a few drops of glacial acetic acid were followed by the addition of acetic anhydride. The appearance of the green color indicates the presence of steroids.

Detection of tannins

Lead Acetate test

Extracts were treated with 10% lead acetate solution; the appearance of white precipitate confirms the presence of tannins.

Ferric Chloride Test

Extracts were treated with 3–4 drops of 1% ferric chloride solution. The appearance of bluish-black color confirms the presence of tannins.

Detection of terpenoids

Salkowski test

Test sample extract (5 ml) was transferred to separate test tubes. 2 ml of chloroform and 3 ml of concentrated sulfuric acid were added to each to form a layer. The formation of a reddish-brown coloration at the interface shows positive results for the presence of terpenoids.

Detection of coumarin

About 0.5 g moistened dry powder of each test sample was taken in separate test tubes. The test tubes were covered with filter paper and presoaked in dilute NaOH. The tubes were kept in a water bath. The filter papers were then exposed to UV light and observed for color development. The appearance of yellowish-green fluorescence indicated the presence of coumarin.

Detection of anthocyanin

Ammonia and 2 ml of 2N HCL were added to 2 ml of aqueous extract. The appearance of pinkish-red color that later turns bluish-violet confirms the presence of anthocyanins.

Quantitative estimation of phytoconstituents

After confirming the presence of phytoconstituents by preliminary phytochemical tests, the aqueous extract of C. zeylanicum was considered for quantitative estimation.

Estimation of total phenolic content

Total phenol content was determined using the method.[5] The amount of phenol in the aqueous extract was determined by Folin–Ciocalteu reagent method. About 2.5 ml of 10% Folin–Ciocalteu reagent and 2 ml of 2% solution of Na2CO3 were added to 1 ml of aqueous extract. The resulting mixture was incubated for 15 min at room temperature. The absorbance of the sample was measured at 765 nm. Gallic acid was used as standard (1 mg/ml). All the tests were performed in triplicates. The results were determined from the standard curve and were expressed as gallic acid equivalents (mg/g of extracted compound).

Estimation of total flavonoid content

Total flavonoid content was estimated following the method.[6] Aluminum chloride colorimetric method was used to determine flavonoid content. About 1 ml of sample was mixed with 3 ml of methanol, 0.2 ml of 10% aluminum chloride, 0.2 ml of 1M potassium acetate, and 5.6 ml of distilled water and kept at room temperature for 30 min. The absorbance was measured at 420 nm. Rutin was used as standard (1 mg/ml). All the tests were performed in triplicates. Flavonoid contents were determined from the standard curve and were expressed as rutin equivalents (mg/g of extracted compound).

Estimation of total saponin content

Total saponin content of C. zeylanicum was determined by following the procedure.[7] To 5 g of samples in each conical flask, 100 ml of aqueous ethanol was added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 55°C. The mixture was filtered and the residue was extracted with another 200 ml of 20% ethanol. The combined extract was reduced to 40 ml over a water bath at about 90°C temperature and the concentrate was transferred into 250 ml separating funnel. Then, 20 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered, while the ether layer was discarded. The purification process was repeated. About 60 ml of n-butanol was added. The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in an oven to constant weight and the saponin content was calculated as a percentage.

Estimation of total tannin content

Total tannin content was estimated by following the method.[8] Quantity of tannins was determined using the spectrophotometric method. About 0.5 g of sample was weighed and taken in a 50-ml plastic bottle, and 50 ml of distilled water was added and stirred for 1 h. The sample was filtered and the volume was made up to 50 ml by adding distilled water. Five ml of filtered sample was then pipetted out into a fresh test tube and mixed with 20 ml of 0.1 ml FeCl3 in 0.1 ml HCl and 0.008M K4Fe (CN) 6 H2O. The absorbance was measured by a spectrophotometer at 395 nm wavelength within 10 min against a suitable blank.

Estimation of total coumarin content

Total coumarin content was estimated by the procedure.[9] A calibration curve was prepared by means of UV–spectrophotometer at 275 nm using the standard compound coumarin (2H-1-benzopyran-2-one). The solvent methanol/water was mixed in the ratio 80:20. For the calibration curve determination, five coumarin standard dilutions were prepared from the stock solution of 1000 μg/ml [Plate 1, [Plate 2], [Plate 3], [Plate 4]. The coumarin value was determined by analyzing the absorbance of an aliquot of 0.8 ml of cinnamon extract diluted into 100 ml of methanol/water (80:20) solution, in a spectrophotometer at 275 nm. The value obtained was used in the line equation revealing the level of coumarin present in the fluid extract.[INLINE:1][INLINE:2][INLINE:3][INLINE:4]

Ferric Reducing Antioxidant power

FRAP method was opted as it was appraised as an authentic method for determining free radical scavenging activity of test samples. In this assay, 1 ml of the test sample in different concentrations was mixed with 1 ml of 0.2 M sodium phosphate ferricyanide in separate test tubes. The reaction mixtures were incubated in a temperature-controlled water bath at 50°C for 20 min, followed by the addition of 1 ml of 10% trichloroacetic acid. The mixtures were then centrifuged for 10 min at room temperature. The supernatant obtained was added to 1 ml of deionized water and 200 μl of 0.1% FeCl3. The blank was prepared in the same manner as the samples except that 1% potassium ferricyanide was replaced by distilled water. The absorbance was measured at 700 nm. The reducing power was expressed as an increase in A700 after blank subtraction.[10]

 Results and Discussion



There is limited research about the bioactive components of different species of cinnamon that are ingested. The conflicting results of cinnamon supplementation trials might be due to the consumption of different cinnamon species with undefined compounds. A thorough understanding about the phytochemicals present in C. zeylanicum and their properties is vital for finding novel functional foods. For any intervention study to have a positive impact on human health, it is necessary to evaluate its efficacy and safety so that it provides the anticipated benefits and is harmless.

Phytochemical screening of cinnamon extracts has revealed the presence of various phytochemicals which have pharmaceutical and industrial values including aromas, dyes, gums, resins, pulp, and fiber with a high bearing on health and commercial sectors. Most of the high value secondary metabolites are produced in scarce amounts by the plant.

[Table 1] illustrates the results of qualitative phytochemical assay. In the present study, preliminary phytochemical assay on the extract of C. zeylanicum bark established the presence of alkaloids, flavonoids, terpenoids, anthocyanins, coumarins tannins, glycosides, polyphenols, and saponins in judicious amounts and indicated the absence of steroids.{Table 1}

C. zeylanicum species contain both water- and oil-soluble compounds and are made of phenyl propanoids, terpenes, flavonoids, and saponins. All these compounds polymerize to form methyl hydroxychalcone polymer which is purported to be the core polymer in lowering blood glucose levels in type 2 diabetes individuals.[11] Saleem et al. 2009[12] in their research has identified bioactive compounds in C. zeylanicum bark such as polyphenols, flavonoids, saponins, tannins and coumarins.

The results of the present study using aqueous extract of C. zeylanicum indicate an abundance of phytochemicals such as flavonoids and polyphenols.

After carrying out the preliminary assay for the presence of phenols, flavonoids, tannins, coumarins, and saponins, the dried aqueous extract powder of C. zeylanicum bark was considered for quantitative estimation.

Quantitative estimation of phytochemicals in Cinnamomum zeylanicum

The quantity of phytochemicals present in the aqueous extract powder of C. zeylanicum is presented in the [Table 2] and [Figure 1], [Figure 2], [Figure 3], [Figure 4].{Table 2}{Figure 1}{Figure 2}{Figure 3}{Figure 4}

The amount of phytochemicals which are found in the aqueous extract was quantitatively determined by standard procedures. The aqueous extract showed different amounts of phytochemicals. On quantification, the total phenol content and total flavonoid content of pulp extract were found to be 436.66 mg/g GAE and 41.92 mg/g QE, followed by saponin which was found to be around 71.25 mg, the tannin content was 43.80 mg. The coumarin content was 57.70 mg. The results were agreeable with the findings of some studies.[13]

Ferric reducing antioxidant power of Cinnamomum zeylanicum

Oxidative stress occurs whenever there is an imbalance between production and accumulation of free radicals. Antioxidants are substances that protect the body from damage caused by free radicals. Antioxidants function as hydrogen donors, metal chelators, or singlet oxygen quenchers.[14] Enzymes such as catalase, glutathione dismutase, and superoxide dismutase present in the body scavenge free radicals.[15] Antioxidants are classified as primary (chain-breaking) antioxidants or secondary (preventive) antioxidants. Phenolic compounds such as phenolic acid, catechins, flavonoids, and phenolic diterpenes are responsible for antioxidant property. These compounds can either absorb or neutralize the effect of free radicals.[16]

The results of the antioxidant investigation of 1120 foods including a wide range of processed foods, fresh fruits, and vegetables were released by the USDA's National Food and Nutrient Analysis Program. This was the largest published systematic screening of antioxidants in dietary items. Ground cloves, dried oregano, ground ginger, ground cinnamon, turmeric powder, dried basil, and ground mustard seed contained >10 mmol antioxidants/100 g. Cassia ranked fourth among the top fifty antioxidant-rich foods.[17]

Ferric reducing antioxidant assay measures the electron-donating capacity of an antioxidant compound. Reduction of ferric to ferrous ion results in the formation of a colored ferrous complex. FRAP assay of C. zeylanicum is illustrated by [Figure 5] and [Figure 6]. The current results are in line with some studies[18] which reported that the secondary metabolites (phytochemicals) are responsible for the antioxidant power of the cinnamon bark which, in turn, makes it an effective therapeutic agent.{Figure 5}{Figure 6}

[Figure 1] and [Figure 2] establish that the sample of AECZ exhibited strong antioxidant property, owing to the presence of high phenolic and flavonoids content. By relating the change of absorbance at 700 nm of the test sample to that of the standard solution of L-ascorbic acid, the results were expressed. The results of Fe 3 + reduction activity indicated the reduction capability of C. zeylanicum. The maximum ferric reducing antioxidant power was found to be 1.377 at 1000 μg. The results of the present study were in accordance with the researchers.[19]

Proanthocyanidins, in cinnamon, have a particularly high hydrophilic, oxygen radical absorbance capacity. Cinnamon bark extracts inhibited the formation of advanced glycation-end products which result in diabetic complications. This inhibition has been attributed to the ability of phenolic compounds in the extracts to trap reactive carbonyl species. Antioxidant variable, ferric reducing antioxidant potential, increased in subjects receiving a cinnamon extract.[20]

Antioxidant studies with C. zeylanicum bark established better free radical scavenging capacity, against free radicals.[21] The antioxidant activity has been attributed to various mechanisms such as prevention of chain initiation, the binding of transition metal ion catalysts, decomposition of peroxides, and the prevention of continued hydrogen abstraction.[22]

A study by Roussel et al.[23] supports that the inclusion of water-soluble C. zeylanicum compounds as antioxidant in the diet could reduce risk factors associated with diabetes and cardiovascular disease.

 Conclusion



The present study clearly highlights certain therapeutic properties of C. zeylanicum. The presence of several unique bioactive compounds present in cinnamon contributes to antioxidant potential. From these findings, it will be possible to discover new natural drugs serving as antioxidant agents for application in the nutritional or pharmaceutical fields in the prevention of free radical-mediated diseases. Furtherin vivo studies are needed to explore their mechanism of action of cinnamon.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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