European Journal of Medicinal Plants

23(1): 1-16, 2018; Article no.EJMP.40565 ISSN: 2231-0894, NLM ID: 101583475

Effect of Brewing Methods and Time on Secondary Metabolites, Total Flavonoid and Phenolic Content of Green and Roasted arabica , and Monsooned Malabar

Mansimran Kaur 1, Shivani Tyagi 2 and Namrata Kundu 1*

1Helix BioGenesis Pvt Ltd, Noida, U.P., India. 2Mewar University, Mewar, Rajasthan, India.

Authors’ contributions

This work was carried out in collaboration between all authors. Author NK designed the study, performed the statistical analysis, wrote the protocol, and first draft of the manuscript. Authors MK and ST managed the analyses of the study. Author MK managed the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/EJMP/2018/40565 Editor(s): (1) Ana Maria Queijeiro Lopez, Professor, Institute of Chemistry and Biotechnology, Federal University of Alagoas, Brazil. (2) Marcello Iriti, Professor, Plant Biology and Pathology, Department of Agricultural and Environmental Sciences, Milan State University, Italy. Reviewers: (1) Rafael Carlos Eloy Dias, Instituto Federal Catarinense, Brazil. (2) Supreena Srisaikham, Burapha University Sakaeo Campus, Thailand. (3) Socaci Sonia, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania. (4) Mary Ann Foglio, Brasil. (5) Esraa Ashraf Ahmed ElHawary, Ain Shams University, Egypt. Complete Peer review History: http://www.sciencedomain.org/review-history/24181

Received 31 st January 2018 Accepted 12 th April 2018 Original Research Article Published 16th April 2018

ABSTRACT

Coffea arabica, Coffea canephora (robusta) and monsooned malabar are the three types of coffee which are well known and used by most of population in India and all over the globe. For consuming coffee various brewing methods are used worldwide, , , Turkish coffee, being the common. Eleven brewing methods are introduced in the paper for brewing of green coffee, some of which are common for brewing tea. Among all the mentioned brewing methods, Decoction method showed the best results, as maximum amount of Flavonoids and Phenols were found to be present in green coffee arabica, values being 69.24 mg QE/g of coffee and 108.67 mg QE/g of coffee respectively, whereas, for ______

*Corresponding author: E-mail: [email protected];

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

robusta it was found to be 47.47 mg QE/g of coffee and 93 mg QE/g of coffee respectively. Brewing time is also considered as one of the major factors for coffee brewing, as if the time is too short, all the flavours will not dissolve and if too long, it may dissolve undesirable components as well. An increasing trend was seen in flavonoid and phenolic content in both arabica (TPC up to 84.11 mg QE/g of coffee) and robusta (TPC up to 78 mg QE/g of coffee) along with monsooned malabar (TPC up to 84.88 mg QE/g of coffee) with increase in brewing time. Another factor considered is the solvent used for brewing. A comparison was made between water and alcohol. Both the , green and roasted showed a significant difference in the values when brewed in ethanol and in water. Results indicate that the brewing methods given may be used along with a variant brewing time depending on its phenolic content. Also, green coffee can be proved a boon with more health benefits as compared to roasted one.

Keywords: Green coffee; roasted coffee; phytochemicals; phenols; antioxidants.

ABBREVIATIONS For many people, especially in western countries, coffee drinking is a part of their TPC : Total Phenolic Content lifestyle and morning habit. Morning cup of coffee TFC : Total Flavonoid Content is a daily routine for millions of people worldwide, QE : Quercetin Equivalent about 40% of the world’s population starts the GAE : Gallic Acid Equivalent day this way [9,10]. Popularity of drinking of a : Absent roasted or infusions is a result of p : Present its stimulating effects, exceptional smell and taste [11].

1. INTRODUCTION The coffee that we know is produced by processing of the green coffee beans in several An evergreen arbour, Coffee, is a member of the stages. Green coffee refers to raw or unroasted Rubiaceae family, is derived from the name of seeds (beans) of Coffea fruits [12-14]. The the province ‘Keffa”, where shepherds from th infusions are believed to accelerate metabolism, Abyssinia/Ethiopia discovered it in 6 century. thus they can be helpful in reducing weight and Since then this beverage is one of the most preventing or overcoming obesity [15]. consumed worldwide due to the pleasant taste, aroma, stimulant effect and health benefits [1-3]. The main constituents of green coffee beans are Coffee plants consist of several species such as carbohydrates (up to 50% of the dry weight), Coffea canephora, , Coffea excels both soluble (galactomannan, arabinogallactan) and Coffea stenophylla [4,5]. The most cultivated and insoluble in water (cellulose), phenolic varieties are (arabica) and species, i.e. , cholorogenic acids, in Coffea canephora (robusta), which are used for addition to polysaccharides, proteins, commercial production, accounting for about polyphenols, melanoids, lipids and minerals [11, 60% and 40% of the world coffee market, 16-,20]. However, the chemical composition and respectively, while Coffea liberica contributes the biological activity of green coffee are highly less than 1% of marketed coffee [6-8]. Arabica affected by roasting [14]. In the process of and robusta may appear similar but there are a roasting chlorogenic acids are particularly number of significant differences between them. degraded as well the content of the phenolic Arabica requires different environmental compounds decrease along with the antioxidant conditions and produces less coffee per hectare activity [21-23]. than robusta, increasing the growth cost. They even differ in caffeine and chlorogenic acid Secondary metabolites from plants have content. Then there is a process that is applied to important biological and pharmacological coffee beans, called monsooned malabar, these activities, such as anti-oxidative, anti-allergic, harvested coffee seeds are exposed to the antibiotic, hypoglycemic and anti-carcinogenic monsoon rain and winds for a period of [24-27]. Largest group of phytochemicals about three to four months, causing the beans to comprises the phenolic chemicals. Plant swell and lose the original acidity, resulting in a polyphenols are secondary metabolites that are flavour profile with a practically neutral pH widely distributed in higher plants [28]. The most balance. important dietary phenolics are phenols acids,

2

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

polyphenols and flavonoids, the latter being the monsooned malabar were bought online from most studied group [29]. The flavonoid subclass Amazon. The roasted arabica beans and roasted of phenols includes minor flavonoids (flavonones robusta beans were from Coorg (Karnataka) and and dihydroflavonols), flavones and flavonols Travancore () respectively. Both were [28]. Flavonoids have a benzo- -pyrone structure freshly roasted on the day before delivery. The and are synthesized by phenylpropanoid monsooned malabar were the homestead spices pathway. Available reports tend to show that from Western Ghats region of Kerala. flavonoids are responsible for the variety of pharmacological activities [30,31], but the best Samples collected were identified and described property of almost every group of authenticated Dr. Ravindra Kumar, Associate flavonoids is their capacity to act as antioxidants. Professor of Plant Biotechnology, Botany The antioxidant activity of flavonoids depends Department, Hindu College, Delhi University, upon the arrangement of functional groups about India. the nuclear structure [32,33]. 2.1.1 Brewing Methods implemented for Brewing techniques and time have a great green coffee impact on the content of polyphenols. The difference in the presence of phytochemical in Green coffee beans of coffee Coffea arabica and different methods depends upon steeping time of Coffea canephora were grounded to make the infusion, type of species, type of solvent and powder in a regular grinder (Philips HL 7720) and temperature [34]. used for all methods except for 10 th i.e. overnight brewing for which coffee beans were directly Although, the brewing time is given in the used. Coffee infusions were prepared by standard coffee brewing techniques used all over following various recipies: the world, the knowledge of extraction behaviour of the main coffee oxidants during time and 1. Soft Infusion: 1 g of coffee powder was various methods might induce to the weighed in weighing balance (High technological factors with major impact on Precision Balance) added to 15 ml of antioxidants extraction. There is no narrative distilled water and kept at 75-80°C in data referring to the content and the effect of the Water bath (Thermo Scientific) for 3-5 method of preparing coffee infusions and time, minutes [35]. on amount of phenolic and antioxidant content. 2. Hard Infusion: 1g of coffee powder was Therefore these parameters can impact not only infused in 15ml of distilled water and kept in coffee brews with higher antioxidant capacity 75-80°C for 25-30 minutes [11]. but also coffee extracts with health properties 3. Ambient Infusion: This method involves that can be used as ingredients in functional infusion of 1 g of coffee powder in 15 ml of foods. Thus, an attempt was made to establish a distilled water at room temperature for 30- correlation among different brewing methods, 40 minutes [34]. brewing time and phenolic content in infusions. 4. Cold Infusion: 1 g of coffee powder was

infused in distilled water at room 1.1 Objective temperature for 15 minutes and then Present study aimed to give various brewing refrigerated at 8-10°C in Refrigerator methods for brewing of green coffee Coffea (Thermo Scientific) for an hour [34]. arabica and Coffea canephora. To give the best 5. Decoction Method: 1 g of coffee powder method among the prescribed brewing methods was added to 15ml of distilled water and on the basis of its total flavonoid and phenolic boiled for 3-4 minutes and then cooled content. Study was also focussed on the down for 30 seconds [34]. importance of brewing time and solvent used for 6. Chilled Green Coffee: 1 g of coffee brewing giving their phytochemical analysis and powder was brewed in distilled boiling phenolic content. water for 2-5 minutes and was then kept in refrigerator at 8-10°C for an hour [34]. 2. MATERIALS AND METHODS 7. Cold Cocktail: In this, 1 g of coffee powder was infused in 40% ethanol for 15 2.1 Collection of Samples minutes and then filtered. Filtered infusion was refrigerated for an hour at 8-10°C [34]. Green and roasted coffee beans of Coffea 8. Hot Cocktail: In this alcoholic infusion, 1 g arabica, Coffea canephora and roasted of coffee powder was added to 40%

3

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

ethanol and kept at room temperature for Turning of upper layer red and yellow 15 minutes followed by heating up to 50- green fluorescence at sulphuric acid layer 55°C [34]. indicates the presence of steroids [38]. 9. Pour-over Method: 1 g of coffee powder 4. Test for Flavonoids: NaOH Test: 2 ml was directly kept on filter paper; hot water 10% NaOH was added to 1 ml of sample. was poured over it and collected. Intense Yellow colour obtained which 10. Overnight Brewing: 10 g of coffee beans changes to colourless on adding 1% HCl were soaked in 150 ml of distilled water indicates the presence of flavonoids [39]. overnight. Mixture was boiled on high 5. Test for Terpenoids: Liebermann – flame. After on boil, it was simmered on Burchard Test: To 1 ml of sample, 2-3 ml low flame for 15 minutes giving occasional of chloroform was added and then equal stir. Mixture was cooled completely and volume of H 2SO 4 was added carefully filtered to remove beans. along the sides to form two different layers. 11. French Press: For this, a press pot or a Formation of reddish-violet colour indicates coffee plunger device (KMX Coffee the presence of Terpenoids [38]. Plunger) was used. The pot was placed on 6. Test for Naphthoquione: To 1 ml of a flat surface, plunger was pulled out and 1 sample, a few drops of 10% KOH were g of coffee powder was added and boiling added. Formation of blue-black colour hot water was gently poured inside. Then indicates presence of Naphthoquione. the plunger was reinserted in to the pot on 7. Test for Inulin: To 1 ml of sample α- the surface of the coffee beverage and napthol and H 2SO 4 solution was added. plunged down after 5 minutes. Once the Formation of brownish colour indicates the press plunger was put down, sample of presence of Inulin. coffee was taken for analysis [11]. 8. Test for Alkaloids: Wagner’s Test: 2 g of Iodine and 6 g of Potassium Iodide were 2.1.2 Preliminary phytochemical screening dissolved in 100 ml of distilled water and for the presence of secondary stored in dark. To 1ml of solvent, 2ml of metabolites the prepared solution was added. Formation of brown and reddish brown Both water and alcohol (ethanol) were used as colour indicates the presence of Alkaloids solvents for the preparation of sample infusions [40]. for screening of phytochemicals. Whereas, time 9. Test for Phenols: Ferric chloride Test: given for brewing was 1, 2, 4, 8 and 16 minutes. To 1 ml of sample 5 ml of distilled water Only infusion with brewing time of 1 min and 16 and 1-2 drops of 1% FeCl 3 were added. A min were screened phytochemically for the red blue-green or purple colour indicates presence and absence of secondary metabolites the presence of Phenols [41]. (both green and roasted coffee) and thereafter Total Phenolic Content (TPC) and Total 2.1.3 Preparation of standard solution Flavonoid Content (TFC) were measured (green coffee). 1 g of Quercetin was dissolved in 100 ml of methanol separately to get 1% solution of 1. Test for Saponins: Foam Test: To 1 ml Quercetin (10 mg/ml) termed as standard (Pipette: Gilson) of brewed sample, 3ml of solution. distilled water was added and shaken vigorously for 2 minutes. Frothing persist 2.1.4 Determination of total flavonoid content on warming indicates the presence of (TFC) saponins [36]. 2. Test for Tannins: Ferric chloride Test: Total flavonoids of the infusions were determined To 1 ml of sample few drops of 1% FeCl 3 by using aluminium chloride spectrophotometric solution were added. Occurrence of blue analysis [42] with slight modifications. 100 µl of black, green or blue green precipitate the samples were raised up to 1 ml and then indicates the presence of tannins [37]. diluted by 2 ml of methanol. Mixture was mixed 3. Test for Steroids: Liebermann – with 0.1 ml of 10% (w/v) aluminium chloride Burchard Test: To 1 ml of sample, 2-3 ml solution and 0.1 ml of 1 M potassium acetate of chloroform was added and then equal solution. The mixture was kept at room volume of H 2SO 4 was added carefully temperature for 30 minutes. Then the maximum along the sides to form two different layers. absorbance was measured at 415 nm using UV-

4

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

Visible spectrophotometer (Hitachi High Phytochemical screening was done to observe Technologies). TFC was calculated from the the presence of secondary metabolites like calibration curve, and the results were expressed saponins, tannins, terpenoids, steroids, as mg of Quercetin equivalent per g weight [43]. napthoquione, inulin, flavonoids, alkaloids and phenols. Initially saponins were found to be 2.1.5 Determination of total phenolic content absent in both green coffee arabica and robusta (TPC) regardless of the solvent in which it was brewed but it was present in roasted robusta and roasted Total phenolics were determined by using Folin- monsooned malabar brewed in water for 1 min Ciocalteau reagent [44] with slight modifications. and 16 min. However roasted robusta and 100 µl of the coffee infusions were made up to roasted malabar in ethanol gave no positive 1ml with distilled water, mixed thoroughly with results for saponins and roasted arabica brewed 2.5ml of 20% (w/v) sodium carbonate for 10 min, for 16 min in water was positive for the same. followed by the addition of 200 µl of Folin- Tannins and Inulin were present in all the Ciocalteau reagent. The mixture was allowed to samples for both the solvents. Steroids were stand further for 30 min in dark at room seen positive in all the brews except green coffee temperature and absorbance was measured at robusta brewed in ethanol for both the times and 750nm using UV-Visible spectrophotometer. TPC also in roasted monsooned malabar brewed in was calculated from the calibration curve, and ethanol for 1 minute. Terpenoids were found to the results were expressed as mg of Quercetin be absent in all the samples with no exceptions. equivalent per g weight [45]. However napthoquione showed an irregular trend as it was completely absent in both green 2.2 Statistical Evaluation coffee arabica and robusta brewed in water and ethanol. It was present in roasted coffee arabica To estimate the accuracy of the experimental brewed for 1 min in water and ethanol but was data, each experiment was performed in absent in 16 - minute brew of both. The roasted triplicates, and the result was expressed as the coffee robusta and roasted malabar brewed for 1 mean ± standard deviation of three replications. and 16 min in water were seen positive for P < 0.05 was considered as statistically napthoquione but the same brewed in ethanol significant. showed no results. Previous studies suggest that tannins, terpenoids, flavonoids and alkaloids 3. RESULTS AND DISCUSSION were present in green coffee, while saponins were absent in the sample under investigation Arabica coffee ( Coffea arabica ) and robusta [48]. coffee ( Coffea canephora ) have different growing conditions and chemical composition, and Alkaloids, flavonoids and phenols were present thereafter organoleptic properties, particularly in all the samples. Infact, green coffee robusta taste and smell, of their beans and infusions are brewed in distilled water showed a double also different [46]. positive for all the three metabolites. Also the yellow colour of the precipitate in the test for Green and roasted coffee Coffea arabica and flavonoids was even more intense in coffees Coffea canephora and roasted monsooned brewed for 16 mins in distilled water than the malabar coffee were brewed for different times ones brewed for 1 minute. This gave us the idea i.e. 1, 2, 4, 8 and 16 minutes in distilled water of increase in flavonoid content with increase in and ethanol. The coffees brewed for 1 minute brewing time. Also same trend was seen in and 16 minute were initially screened phenols. Therefore, we further subjected all the phytochemically to detect the presence and coffees i.e. green and roasted coffee arabica and absence of different secondary metabolites. robusta along with roasted monsooned malabar for quantitative analysis of TFC and TPC Phytochemicals are various active compounds (Table 2- Table 11). found in plants which have been used in a variety of industrial as well as commercial applications, In plants, there are about 8000 known phenolic since synthetic compounds do have side effects. compounds with aromatic ring produced as Phytochemicals of plants show different secondary metabolites. They protect the plant biological activities, which play an important against pathogens and abiotic stress such as role in the protection against chronic diseases changes in temperature, water content, exposure [47]. to UV light and deficiency of mineral nutrients.

5

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

The level of individual phenolic compounds content with decoction method having the normally depends on the maturity of the beans maximum of 108.67 mg QE/g of coffee, chilled and a smaller degree, on the composition of the green coffee being the next having 101 mg QE/g soil, climatic conditions and agricultural practices of coffee and pour over method having the related to the coffee bush [17,49]. lowest of value 72.78 mg QE/g of coffee (Table 1 and Fig. 2). Total flavonoids content and total phenolic content was determined by spectrophotometric Same brewing methods were performed for analysis using aluminium chloride and sodium green coffee robusta, but the values seen were carbonate respectively. Quercetin was used as quite different from green coffee arabica, the best the standard for both (Figs. 5 and 6). being overnight brewing method with value of 57 mg QE/g of coffee. Next value being 53.23 mg Different brewing methods were performed for QE/g of coffee for hard infusion method. green coffee arabica and robusta . These However lowest flavonoid content was seen in infusions were then subjected to determine the cold cocktail method i.e. 10.35 mg QE/g of coffee total flavonoids and phenols (Figs. 1 and 2). and the pour over method being very close to it For green coffee arabica, the decoction method with value 10.59 mg QE/g of coffee. The phenolic was reported to have the maximum flavonoid content was obviously more than the flavonoid content of 69.24 mg QE/g of coffee, second content but had a different trend. The overnight being the chilled green coffee of value 61.65mg brewing method and chilled green coffee had QE/g of coffee. Lowest being the pour over similar value of 93.33 mg QE/g of coffee. There method i.e. 7.53 mg QE/g of coffee. Rest of the was a slight difference in the values of cold methods lying in between these two were having cocktail method and pour over method i.e. 71.89 flavonoid content in the range of 14-36 mg QE/g mg QE/g and 71 mg QE/g of coffee respectively of coffee. Similar trend was seen in the phenolic (Table 1 and Fig. 2).

Table 1. TFC and TPC of various brewing methods implemented in green coffee Coffea arabica and Coffea canephora

Brewing methods Total flavonoid content Total phenolic content (mg QE / g of coffee) (mg QE / g of coffee) Arabica Robusta Arabica Robusta Soft Infusion 27.18 27.65 94 78.33 Hard Infusion 35.76 53.23 96 89.11 Ambient Infusion 27.47 21.3 93 77 Cold Infusion 27.53 24.76 89.78 77.44 Decoction method 69.24 47.47 108.67 93 Chilled Green Coffee 61.65 45.24 101 93.33 Cold Cocktail 13.59 10.35 83.56 71.89 Hot Cocktail 25.77 11.65 92.44 73.78 Pour over method 7.53 10.59 72.78 71 Overnight brewing 22.06 57 88.67 93.33 French Press 29.94 18.18 100.78 74.11

(1) (2)

Fig. 1. (1) and (2) Representing the green coffee extracts of arabica prepared after all the eleven brewing methods implemented

6

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no. EJMP.40565

TFC and TPC for different Brewing Methods in Green Coffee Coffea arabica and Coffes canephora 120 100 80

60 Total Flavonoid (mg QE / g of coffee Arabica 40 Total Flavonoid (mg QE / g of 20 coffee Robusta 0 Total Phenolic (mg QE / g of mg QE/ mg g ofextract ofcoffee coffee Arabica Total Phenolic (mg QE / g of coffee Robusta

Brewing methods implemented

Fig. 2. TFC and TPC in various brewing methods followed for Coffea arabica and Coffea canephora

It is important that the brewing time must be The amount of time that the water is in contact exactly controlled. Improper brewing time can be with the coffee grounds is another important one of the reasons that people get different flavour factor. In a drip system, the contact time results while preparing coffee. If the extraction should be approximately 5 minutes. If one is time is shortened, we'll fail to dissolve the making your coffee using a French Press, the essential flavour compounds. Again, over - contact time should be 2-4 minutes. Espresso extraction of the same will dissolve too many of has an especially brief brew time - the coffee is in the undesirable compounds [50]. contact with the water for only 20 -30 seconds. Cold brew, on the other hand, should steep Table 2. Results showing Phytochemical overnight (about 12 hours) [51]. screening for the presence of mentioned secondary metabolites of green coffee Table 3. Results showing phytochemical arabica brewed in disti lled water for 1 and 16 screening for the presence of mentioned minutes secondary metabolites of green coffee arabica brewed in ethanol for 1 and 16 Secondary metabolite 1 min 16 min minutes Saponins a a Tannins p p Secondary metabolite 1 min 16 min Steroids p p Saponins a a Flavonoids p p Tannins p p Terpenoids a a Steroids p p Napthoquione a a Flavonoids p p Inulin p p Terpenoids a a Alkaloids p p Napthoquione a a Phenols p p Inulin a p Alkaloids p p There is important exception to "exact brewing Phenols p p time". About 90% of the solubles are extracted during the early phase of the brewing process After different brewing methods for green coffee [50]. arabica and robusta, all the coffees including

7

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

green and roasted coffee arabica, robusta and An increasing trend was seen in the flavonoid roasted monsooned Malabar were brewed for 1, and phenolic content and with increase in time of 2, 4, 8 and 16 minutes in distilled water and brewing with distilled water. However, brewing in ethanol and then subjected for quantification of ethanol showed different results. It seemed to flavonoids and phenols. increase first and then started decreasing, with 4 min brewing showing the maximum value in case Table 4. Results showing phytochemical of roasted coffee, but for green coffee the trend screening for the presence of mentioned was same as observed with distilled water. secondary metabolites of green coffee robusta brewed in distilled water for 1 and 16 Table 7. Results showing phytochemical minutes screening for the presence of mentioned secondary metabolites of roasted coffee Secondary metabolite 1 min 16 min arabica brewed in ethanol for 1 and 16 Saponins a a minutes Tannins p p Steroids p p Secondary metabolite 1 min 16 min Flavonoids p p Saponins p a Terpenoids a a Tannins p p Napthoquione a a Steroids p p Inulin p p Flavonoids p p Alkaloids p p Terpenoids a a Phenols p p Napthoquione p a Inulin p p Table 5. Results showing phytochemical Alkaloids p p screening for the presence of mentioned Phenols p p secondary metabolites of green coffee robusta brewed in ethanol for 1 and 16 Table 8. Results showing phytochemical minutes screening for the presence of mentioned secondary metabolites of roasted coffee Secondary metabolite 1 min 16 min robusta brewed in distilled water for 1 and 16 Saponins a a minutes

Tannins p p Secondary metabolite 1 min 16 min Steroids a a Saponins p p Flavonoids p p Tannins p p Terpenoids a a Steroids p p Napthoquione a a Flavonoids p p Inulin p p Terpenoids a a Alkaloids p p Napthoquione p p Phenols p p Inulin p p Alkaloids p p Table 6. Results showing phytochemical Phenols p p screening for the presence of mentioned secondary metabolites of roasted coffee TPC and TFC might be related with antioxidant arabica brewed in distilled water for 1 and 16 activity [52]. Green coffee arabica exhibited more minutes amounts of flavonoids and phenols than green coffee robusta . However roasted coffee robusta Secondary metabolite 1 min 16 min showed more amounts of polyphenols than Saponins p a roasted coffee arabica for brewing in distilled Tannins p p water, while reverse in case of brewing with Steroids p p ethanol as a solvent. As far as green and roasted Flavonoids p p coffee of the same species is compared, roasted Terpenoids a a coffee gave the presence of more phenols as Napthoquione p a compared to green, when brewed in distilled Inulin p p water. But, using ethanol as a brewing solvent Alkaloids p p showed more presence of phenols in case of Phenols p p green than in roasted.

8

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

Table 9. Results showing phytochemical 19.5 to 35.1 mg QE/g of coffee and 82 to 88 mg screening for the presence of mentioned QE/g of coffee respectively as compared to secondary metabolites of roasted coffee green coffee, where both TFC and TPC robusta brewed in ethanol for 1 and 16 increased with brewing time i.e. 18.9 to 40.1 mg minutes. QE/g of coffee and 71.6 to 83.4 mg QE/g of coffee respectively. When brewed in ethanol for Secondary metabolite 1 min 16 min the given time intervals, both flavonoid and Saponins a a phenolics were found to be more in green coffee Tannins p p then in roasted coffee. Flavonoids in green and Steroids p p roasted coffee when brewed in ethanol were Flavonoids p p increased from 10.9 to 14.7 mg QE/g of coffee Terpenoids a a and 3.8 to 8.2 mg QE/g of coffee respectively. Napthoquione a a Phenolic content for green coffee was found to Inulin p p be maximum when brewed for 2 mins, and Alkaloids p p afterwards it decreased to 79.8 mg QE/g of Phenols p p coffee and then it was found to be 84.1 mg QE/g of coffee. Therefore, no consistent trend was Flavonoids content varied from 11.9 to 34.5 mg observed. In case as roasted arabica coffee, the QE/g of coffee and 21.1 to 35.3 mg QE/g of phenolic content was found to be increase from 1 coffee for green and roasted coffee Coffea min to 4 min i.e. 66 to 71.7 mg QE/g of coffee, canephora respectively (Table 10 and 15) (Fig. 8 and then it decreased to 63.3 mg QE/g coffee and 10), when brewed in distilled water. Same with increase in time to 16 mins (Table 14 and trend was observed for the phenolic content i.e. 16) (Figs. 9 and 11). 62.8 to 78 mg QE/g of coffee and 83 to 92.5 mg QE/g of coffee for green and roasted robusta Table 10. Results showing phytochemical coffee respectively. When solvent was changed screening for the presence of mentioned from distilled water to alcohol, the trend in secondary metabolites of roasted coffee flavonoid and phenolic content was reversed. monsooned malabar brewed in distilled water TFC varied from 6.5 to 10.1 mg QE/g of coffee for 1 and 16 minutes for green coffee robusta and 3.6 to 7.6 mg QE/g of coffee for roasted coffee robusta. In the Secondary Metabolite 1 min 16 min present study, TPC was found to be present in Saponins p p more amounts and increased with time in green Tannins p p coffee robusta than roasted coffee robusta when Steroids p p brewed in alcohol as a solvent i.e. 63.3 to 68.9 Flavonoids p p mg QE/g of coffee. Whereas, for roasted coffee Terpenoids a a robusta, a decreasing trend was found for Napthoquione p p brewing in ethanol i.e. 64.6 to 55.5 mg QE/g of Inulin p p coffee which was quite different from TPC in Alkaloids p p ethanolic extract of arabica green coffee from Phenols p P Toraja, Lintong, and Mandailing were 28.31, 13.74, and 70.55 g GAE/100 g [52]. In another Table 11. Results showing phytochemical study, it was reported that TPC in arabica coffee screening for the presence of mentioned which were extracted using isopropanol-water secondary metabolites of roasted coffee (80:20), (70:30), (60:40) were 23.29, 28.71, monsooned malabar brewed in ethanol for 1 32.19% GAE, while TPC in robusta coffee were and 16 minutes 22.89, 26.19, 31.71% GAE. TFC in the present study demonstrated that ethanolic arabica coffee Secondary metabolite 1 min 16 min extract of Toraja, Lintong and Mandailing were Saponins a a 3.57, 3.60, 5.86 g QE/100 g and their TCC 0.17, Tannins p p 0.28, 0.24 g BE/100 g respectively [53]. Steroids a p Flavonoids p p As far as Coffea arabica was kept into Terpenoids a a consideration, initially the brewing trend with Napthoquione a a distilled water was same as seen in Coffea Inulin p p canephora . Both TFC and TPC were found to be Alkaloids p p present more for roasted coffee arabica, being Phenols p p

9

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

Table 12. TFC and TPC variation in roasted brewed in ethanol- 3.5 to 9.2 mg QE/g of coffee. coffee monsooned malabar with time and But as phenols are considered when brewed in solvents. alcohol, the content increased till 4 min brewing – from 60 to 65.6 mg QE/g coffee and then it Time Water Ethanol decreased till 16 min - 61.7 mg QE/g of coffee TFC TPC TFC TPC (Table 12 and Fig. 7). 1 min 17.11 71.55 3.58 60 2 min 20.76 71.88 4.82 64.66 Table 13. TFC and TPC variation in green 4 min 21.52 73.33 5 65.66 coffee robusta with time and solvents 8 min 25.58 84.55 7 64.55 16 min 35.35 84.88 9.23 61.77 Time Water Ethanol Units: mg QE/g of coffee TFC TPC TFC TPC 1 min 11.89 62.78 6.41 63.33 For monsooned malabar, total flavonoids and 2 min 13.35 63.89 7.3 70.78 phenolics increased with time when brewed in 4 min 24.41 74.11 20.41 77.33 distilled water i.e. 17.1 to 35.3 mg QE/g of coffee 8 min 33.18 77.22 13.23 68.22 and 71.5 to 84.8 mg QE/g of coffee respectively. 16 min 34.47 78 10.12 68.89 Same trend was observed for flavonoids when Units: mg QE/g of coffee

(3) (4)

Fig. 3. (3) and (4) representing the green coffee extracts of robusta prepared after all the eleven brewing methods implemented.

Fig. 4. Showing coffee extracts prepared after brewing for 1, 2, 4, 8 and 16 minutes of green coffee arabica in ethanol and distilled water

10

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no. EJMP.40565

TFC Standard of Quercetin 2 1.8 y = 0.0174x 1.6 R² = 0.9851 1.4 1.2 1 0.8 OD @ 417 0.6 Linear (OD @ 417) 0.4 Absorbance417 at nm 0.2 0 0 50 100 150 Volume of Quercetin in µl

Fig. 5. Standard for TFC

TPC Standard of Quercetin 1 0.9 y = 0.0094x 0.8 R² = 0.1322 0.7 0.6 0.5 0.4 OD @ 760 0.3 Linear (OD @ 760) 0.2 Absorbance at760nm 0.1 0 0 50 100 150 Volume of Quercetin in µl

Fig. 6. Standard for TPC

Table 14. TFC and TPC variation in green Table 15. TFC and TPC variation in roasted coffee arabica with time and solvents coffee robusta with time and solvents

Time Water Ethanol Time Water Ethanol TFC TPC TFC TPC TFC TPC TFC TPC 1 min 18.94 71.67 10.94 76.56 1 min 21.11 83 3.64 64.66 2 min 22.47 79 14.3 87.56 2 min 26.35 88.22 5.23 64.44 4 min 32.76 82 19.35 83.89 4 min 31.52 89.88 6.29 63.55 8 min 37.53 83 17.23 79.89 8 min 33.47 90.22 7.47 60.88 16 min 40.18 83.44 14.65 84.11 16 min 35.35 92.55 7.64 55.55 Units: mg QE/g of coffee Units: mg QE/g of coffee

11

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no. EJMP.40565

Variation in TFC & TPC of Monsooned Malabar with time and solvents 90 84.55 84.88 80 71.55 71.88 73.33 64.66 65.66 64.55 70 60 61.77 60 50 Water TFC 40 35.35 25.58 Water TPC 30 20.76 21.52 17.11 Ethanol TFC 20 9.23 mg QE/g mg ofCoffee 4.82 5 7 10 3.58 Ethanol TPC 0 1 min 2 min 4 min 8 min 16 min Brewing time

Fig. 7. TFC and TPC variation in roasted coffee monsooned malabar with time and solvents

Variation in TFC & TPC of Green coffee Robusta with time and solvents 90 74.1177.33 77.22 78 80 70.78 68.22 68.89 70 62.7863.33 63.89 60 50 Water TFC 33.18 34.47 40 Water TPC 30 24.4120.41 11.89 13.35 13.23 Ethanol TFC 20 7.3 10.12 mg mg QE/g coffee of 6.41 10 Ethanol TPC 0 1 min 2 min 4 min 8 min 16 min Brewing time

Fig. 8. TFC and TPC variation in green coffee robusta with time and solvents

Variation in TFC & TPC of Green coffee Arabica with time and solvents 100 87.56 90 82 83.89 83 83.4484.11 76.56 79 79.89 80 71.67 70 60 Water TFC 50 37.53 40.18 40 32.76 Water TPC 30 18.94 22.47 19.35 14.3 17.23 14.65 Ethanol TFC

mg QE/g mg ofcoffee 20 10.94 10 Ethanol TPC 0 1 min 2 min 4 min 8 min 16 min Brewing Time

Fig. 9. TFC and TPC variation in green coffee arabica with time an d solvents

12

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no. EJMP.40565

Variation in TFC & TPC of Raosted coffee Robusta with time and solvents 100 92.55 88.22 89.88 90.22 90 83 80 70 64.66 64.44 63.55 60.88 60 55.55 Water TFC 50 40 31.52 33.47 35.35 Water TPC 26.35 30 21.11 Ethanol TFC

mg mg QE/g ofcoffee 20 6.29 7.47 7.64 Ethanol TPC 10 3.64 5.23 0 1 min 2 min 4 min 8 min 16 min Brewing time

Fig. 10. TFC and TPC variation in roasted coffee robusta with time and solvents

Variation in TFC & TPC of Roasted coffee Arabica with time and solvents 100 86.77 88 90 82 84.33 85.77 80 69.22 71.77 70 66 63.77 63.33 60 Water TFC 50 40 32.64 33.88 35.11 Water TPC 24.41 30 19.52 Ethanol TFC mg QE/g mg ofcoffee 20 6.23 7.52 8.29 Ethanol TPC 10 3.88 4.76 0 1 min 2 min 4 min 8 min 16 min Brewing time

Fig. 11. TFC and TPC variation in roasted coffee arabica with time and solvents

Table 16. TFC and TPC variation in roasted 4. CONCLUSION coffee arabica with time and solvent s We, the authors reach a conclusion that instead Time Water Ethanol of using the general brewing methods that are TFC TPC TFC TPC used till date, the mentioned methods in the paper can also be used for brewing coffee 1 min 19.52 82 3.88 66 according to taste. Moving towards brewing time, 2 min 24.41 84.33 4.76 69.22 it may be increased according to purpose for 4 min 32.64 85.77 6.23 71.77 which one is consuming coffee for. Apart from 8 min 33.88 86.77 7.52 63.77 that, we would like to conclude that alcohol may 16 min 35.11 88 8.29 63.33 also be used as a solvent for brewing for both Units: mg QE/g of coffee green and roasted coffee.

13

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

COMPETING INTERESTS antioxidant activity of green coffee brews. Food Chem. 2015;182:302-308. Authors have declared that no competing 12. Farah A, dos Santos TF. The coffee plant interests exist. and beans: An introduction. In V.R. Preedy (Ed.). Coffee in health and disease REFERENCES prevention. Elsevier Inc: Academic Press. 2015;5-10. 1. Saura-Calixto F, Goni I. Antioxidant 13. Valentin J, Watling RJ. Provenance capacity of the Spanish Mediterranean establishment of coffee using solution ICP- diet. Food Chem. 2006;94:442-447. MS and ICP-AES. Food Chem. 2013;141: 2. Monente C, Ludwig IA, Irigoyen A, de 98-104. Pena MP, Cid C. Assessment of total (free 14. Sevcan S, Selda M, Murat Y, Munevver A. and bound) phenolic compounds in spent Elemental composition of green coffee and coffee extracts. J Agri Food Chem. its contribution to dietary intake. Food 2015;63:4327-4334. Chem. 2017;215:92-100. 3. Gebeyehu T, Bikila SL. Determination 15. Shimoda H, Seki E, Aitani M. Inhibitory of caffeine content and antioxidant effect of green extract on fat activity of coffee. Am J App Chem. accumulation and body weight gain in 2015;3:69-76. mice. BMC Complementary and Alt Med. 4. AC Charrier, J Berthaund. Botanical 2006;6:9. classification of coffee. Coffee: Botany, 16. Bicchi CP, Binello AE, Pellegrho GM, Biochemistry and Production of Beans and Vanni AC. Characterization of green and Beverage. M.N. Clifford and K.C. Willson, roasted coffees through the chlorogenic Eds., Croom Helm, London, UK, 1985;13- acid fraction by HPLC-UV and principal 47. component analysis. J of Agri and Food 5. A Farah and TF Dos Santo. The coffee Chem. 1995;43:1549-1555. 17. Chu YF. Coffee: Emerging health effects plant and beans. Coffee in Health and st Disease Prevention. Academic Press, New and disease prevention (1 ed.) Oxford: York, NY, USA. 2015;5-10. Wiley – Blackwell; 2012. 6. Rodrigues NP, de Jesus Garcia Salva T, 18. Fischer M, Reimann S, Trovato V, Bragagnolo N. Influence of influence of Rodgwell RJ. Polysaccharides of green coffee genotype on bioactive compounds arabica and robusta coffee beans. and the in vitro capacity to scavenge Carbohydrate Res. 2001;330:93-101. reactive oxygen and nitrogen species. J 19. Naidu MM, Sulochanamma G, Sampathu Agri Food Chem. 2015;63:4815-4826. SR and Srinivas P. Studies on extraction 7. International coffee organization. Coffee and antioxidant potential of green coffee. Market Report; 2014. Food Chem. 2008;107:377-384. 20. Wei F, Furihata K, Koda M, Hu F, Kato R, 8. Schievano E, Finotello C, de Angelis E, 13 Mammi S, Navarini L. Rapid authentication Miyakawa T. C – NMR based of coffee blends and quantification of 16-O- Metabolomics for the classification of methylcafestol in roasted coffee beans by green coffee beans according to variety nuclear magnetic resonance. J Agri Food and origin. J of Agri and Food Chem. 2012; Chem. 2014;62:12309-12314. 60:10118-10125. 9. Mussatto SI, Machado EMS, Martins S, 21. Brezova V, Slebodova A; Stasko A. Coffee Teixeira JA. Production, consumption and as a source of antioxidants: An EPR study. application of coffee and its industrial Food Chem. 2009;114:859-868. residues. Food Bioprocess Tech. 2011;4: 22. Koseoglu YP, Hacibekiroglu I, Kolak U. 661-672. Effect of roasting on antioxidant and 10. Oliveira M, Casal S, Morais S, Alves C, anticholinesterase capacities of coffee. J of Dias F, Ramos S, Mendes E, Delerue- Food and Nut Res. 2014;53:232-239. Matos C, Oliveira BPP. Intra- and 23. Wei F, Tanokura M. Chemical changes in interspecific mineral composition variability the components of coffee beans during of commercial coffees and . In V.R. Preedy (Ed.). Coffee in substitutes. Contribution to mineral intake. health and disease prevention. Elsevier Food Chem. 2012;130:702-709. Inc: Academic Press. 2015;83-91. ć 11. Ewelina S, Pwael P, Anna SM. The 24. Milan S. Stankovi . Total phenolic content, content of Ca, Cu, Fe, Mg and Mn and Flavonoid concentration and antioxidant

14

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

activity of Marrubium peregrinum L. Padmanaban N, Krishnan MRV. extracts. Kragujevac J of Science. 2011; Phytochemical investigation on a tropical 33:63-72. plant. Pak J of Nut. 2009;8:83-85. 25. Borneo R, Leon EA, Aguirre A, Ribotta P, 37. Treare GE, Evans WC. Pharmacognosy Cantero JJ. Antioxidant capacity of 17 th edition, Bahive Tinal, London. 1985; medicinal plants from the Province of 149. Cordoba (Argentina) and their in vitro 38. Gibbs RD. Chemotaxonomy of flowering testing in model food system. Food Chem. plants. McGill Queen’s University Press, 2008;112:664-670. Montreal and London. 1974;1. 26. Katalinic V, Milos M, Kulisic T, Jukic M. 39. Ajayi IA, Ajibade O, Oderinde RA. Screening of 70 medicinal plant extracts Preliminary phytochemical analysis of for antioxidant capacity and total phenols. some plant seeds. Res J of Plant Sci. Food Chem. 2004;94:550-557. 2011; 1,3. 27. Mulabagal V, Tsay H. Plant cell cultures - 40. Geetha TS, Geetha N. Phytochemical an alternative and efficient source for the screening, quantitative analysis of primary production of biologically important and secondary metabolites of secondary metabolites. Int J App Sci and Cymbopogan citrates (DC) stapf. Leaves Engg and Tech. 2004;2:29-48. from Kodaikanal hills, Tamilnadu. Int J of 28. Cora JD, J Bruce G. Phytochemicals: Pharma Tech Res. 2014;6(2):521-529. Neutraceuticals and human health. J of the 41. Devi PS, Satyanarayana B, Naidu MT. Sci of Food and Agri. 2000;80:1744-1756. Phytochemical screening for secondary 29. King A and Young G. Characteristics and metabolites in Boswellia serrata Roxb. and occurrence of phenolic phytochemicals. J Wrightia tinctoria (Roxb.) R.Br. Notulae Am Diet Assoc. 1999;99:213-218. Scientia Biologicae. 2014;6(4):474-477. 30. Mahomoodally MF, Gurib-Fakim A, 42. Chang CC, Yang MH, Wen HM. Estimation Subratty AH. Antimicrobial activities and of total flavonoid content in propolis by two phytochemical profiles of endemic complementary colorimetric methods. J of medicinal plants of Mauritius. Pharma Bio. Food Drug Analysis. 2002;10:178-182. 2005;43(3):237-242. 43. Quy DD, Artik EA, Phuong LN, Lein HH, 31. Pandey AK. Anti-staphylococcal activity of Felycia ES, Suryadi I, Yi-Hsu J. Effect of a pan-tropical aggressive and obnoxious extraction solvent on total phenol content, weed Parihenium histerophorus : An in vitro total flavonoids content and antioxidant study. National Academy Science Letters. activity of Limnophila aromatic. J of Food 2007;30:11-12,383–386. and Drug Analysis. 2013;1-7. 32. Kelly EH, Anthony RT, and Dennis JB. 44. Singleton VL, Joseph AR. Colorimetry of Flavonoid antioxidants: Chemistry, total phenolics with phosphomolybdic - metabolism and structure-activity phosphotungstic acid reagents. Am J of relationships. J of Nut Biochem. 2002; Enology and Viticulture. 1965;16:144-158. 13(10):572–584. 45. Kaur C and Kapoor HC. Anti-oxidant 33. Pandey AK, Mishra AK, Mishra A. activity and total phenolic content of some Antifungal and antioxidative potential of oil Asian vegetables. Int J of Food Sci and and extracts derived from leaves of Indian Tech. 2002;37:153-161. spice plant Cinnamomum tamala. Cellular 46. Vignoli JA, Viegas MC, Bassoli DG and de and Mol Bio. 2012;58:142–147. Benassi T. Roasting process affects 34. Safdar N, Sarfaraz A, Kazmi Z and Yasmin differently the bioactive compounds and A. Ten different brewing methods of green the antioxidant activity of arabica and tea: comparative antioxidant study. J of robusta coffees. Food Res Inter J. 2014; App Bio Biotech. 2016;4(03):033-040. 61:279-285. 35. Wolska J, Janda K, Jakubezyk K, 47. Oloyed OI. Chemical profile of unripe pulp Szymkowiak M, Chlubek D, Gutowska I. of Carica pagaya . Pak J of Nut. 2005;4: Levels of antioxidant activity and fluoride 379-381. content in coffee infusions of Arabica, 48. Raoof GFA, Mohamed KY, Mohammed Robusta and Green coffee beans in HM. Phytochemical evaluation, accordance to their brewing methods. Bio anti-obesity and antihyperlipidemic effects of Trace Elements Res. 2017;179:327-333. of combined administration of green 36. Kumar A, Ilavarasn R, Jayachandran T, coffee. Cinnamon and Ginger. Plant. 2017; Decaraman M, Aravindhan P, 5(5):80-84.

15

Kaur et al.; EJMP, 23(1): 1-16, 2018; Article no.EJMP.40565

49. Farah A, Donangelo CM. Phenolic three regions using ABTS and DPPH compounds in coffee. Braz J of Plant asaays. Asian J of Pharma and Clinical Physio. 2006;18:23-36. Res. 2016;9(2):189-193. 50. Available:https://blackbearcoffee.com/reso 53. Naidu MM, Sulochanamma G, Sampathu urces/87 SR, Srinivas P. Studies on extraction 51. Available:http://www.ncausa.org/About- and antioxidant potential of green Coffee/How-to-Brew-Coffee coffee. Food Chem. 2008;107: 52. Irda F, Annisa, Komar R. Antioxidants 377-384. activities of Arabica green coffee from ______© 2018 Kaur et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history/24181

16