Quantitative Analysis of Caffeine Content from Coffee Growing in Different Regions of Rwanda

QUANTITATIVE ANALYSIS OF CAFFEINE CONTENT FROM COFFEE GROWING IN DIFFERENT REGIONS OF RWANDA.

1 2 3 Barasikina Jean d’Amour , Kagiraneza Olivier Placide , NSHOGOZ Gilbert

1University of Rwanda /College of Science and Technology, Department of Chemistry, POBX: 3900 Kigali-Rwanda 2Eduardo Mondlane University, Department of Chemical Engineering, postal 257 Maputo- Mozambique 3lecturer at University of Rwanda-College of Science and Technology.

ABSTRACT Coffea is considered as a large genus (with more than 90 species) of flowering plants belongs in the family Rubiaceae. However, coffee ranks as one of the major commodity crops in the world and is the essential export product of some countries. Moreover, coffee beans found on the market are produced from two different species of the Coffea genus such as Coffea arabica and Coffea canephora syn. Coffea robusta . Nowadays, due to its pleasant taste, aroma, stimulant effect, and health benefits, coffee is one among the most widely consumed beverages throughout the world includes Rwanda country. Both species of coffee present a rich source of biologically active compounds such as caffeine. Caffeine is defined as a xanthine alkaloid compound that acts as a stimulant in humans. It is normally found in the leaves and beans of the coffee plant. In addition to that, caffeine is widely used to enhance alertness and improve performance as it acts as psychostimulant. Consequently, the strong pharmacological effects of caffeine have led to consumer demand for caffeine- free coffee beverages. Due to the high consumption rate of caffeine and its potential physiological effects, both health professionals and consumers need to know the exact caffeine content in food. The way to assess their content to find a more precise relationship between the amounts of consumed caffeine and it’s physiological effects ,it is therefore important to precisely determine the caffeine content in different coffee types. The research’s objective was to determine Caffeine content in coffee growing in different Regions of Rwanda in order to identify the coffee region that contains the highest amount of caffeine. The Coffee samples presenting four growing regions of Rwanda have been taken from NAEB and brought to the College of Science and Technology (CST) chemistry department Laboratory. Quantitative analysis of Caffeine was performed by using a UV-Visible spectrometer (UV-Vis spectrometer) at 650 nm. The results showed that the highest caffeine concentration was obtained from coffee grown in the Western province region presented by GASHONGA Coffee (RUSIZI District) with 9.4±1.962 ppm of caffeine concentration. This was followed by coffee grown in the Southern province region presented by MARABA Coffee (HUYE District) which contains 7.5333±0.8885 ppm of caffeine concentration. The third coffee in caffeine concentration was MUSASA Coffee (GAKENKE District) grown in the Northern Province region which contains 3.8±1.270ppm of caffeine. The last caffeine concentration was obtained in coffee from Eastern province, presented by HUMURE Coffee (GATSIBO District) with 3.2667± 1.578ppm of caffeine Western province region presented by GASHONGA Coffee (RUSIZI district). Keyword: Coffee; Caffeine; alkaloid; stimulant effects; pharmacological effects 1. INTRODUCTION family Rubiaceae. However, coffee ranks as one of the major commodity crops in the world and is the

1.1. Background essential export product of some countries. Coffee

Coffea is considered as a large genus (with more beans are widely cultivated in tropical countries than 90 species) of flowering plants belongs in the in plantations for both local consumption and

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export to temperate countries. Coffee ranks as one dioxopurine. Thus pure anhydrous caffeine is a of the world's major commodity crops and is the powder with white odorless and it has a melting major export product of some countries [1] point of 235–238 °C. At room temperature, caffeine is moderately soluble in water (2 g/100

Coffee beans purchased are produced from two mL), but it is more soluble in boiling water (66 different species of the Coffea genus: Coffea g/100 mL). It is also moderately soluble in ethanol arabica and Coffea canephora syn. Coffea robusta. (1.5 g/100 mL). It is weakly basic (pKa = ~0.6) Traditionally, C. canephora, primarily from African requiring strong acid to protonate it. However, origins, was the dominant component in most caffeine does not contain any stereogenic centers coffee blends available in Belgium/Luxemburg, and hence is classified as an achiral molecule. France, Portugal, and the UK, while most blends available in Scandinavia, Austria, Switzerland, Moreover, the xanthine core of caffeine contains Germany, Italy, and Spain incorporated a much two fused rings, pyrimidinedione, and imidazole. higher proportion of C. Arabica[2]. Wherever, the pyrimidinedione in turn contains

two amide functional groups that exist Nowadays, due to its pleasant taste, aroma, predominately in a zwitterionic resonance form stimulant effect, and health benefits, coffee is one where the nitrogen atoms are double bonded to among the most widely consumed beverages their adjacent amide carbons atoms. Hence all six throughout the world includes Rwanda country. of the atoms within the pyrimidinedione ring Both species of coffee present a rich source of system are sp2 hybridized and planar. Therefore biologically active compounds such as caffeine[3]. the fused 5,6 ring core of caffeine contains a total

Caffeine is defined as a xanthine alkaloid of ten pi electrons and hence according to Hückel's compound that acts as a stimulant in humans. It is rule is aromatic[6]. normally found in the leaves and beans of the coffee plant. In addition to that, caffeine is widely used to enhance alertness and improve performance as it acts as psychostimulant [4]. 1.2. Caffeine properties Generally, caffeine belongs among the family of heterocyclic compounds known as purines. Its systematic name is 3, 7-dihydro-1, 3, 7-trimethyl- 1H-purine-2, 6-dione; it is also known as 1, 3, 7- Figure 1.structure of caffeine trimethylxanthine, and 1, 3, 7-trimethyl-2, 6- Table 1.Properties of Caffeine General Systematic name 3,7-dihydro-1,3,7-trimethyl-1H-Purine-2,6-dione Other names 1,3, 7-Trimetylxanthine, Trimethylxanthine, Methyl theobromine.

Molecular formula C8H10N4O2 SMILES O=C1C2=C(N=CN2C)N(C(=O)N1C)C Molar mass 194.19 g molâˆ’1 Appearance Odorless, white needles or powder

1.3. Biosynthesis of Caffeine then theobromine, later being the penultimate precursor of caffeine synthesis. It is therefore, to

Caffeine is normally synthesized in plants from the be readily available as a byproduct of purine nucleotides AMP, GMP, and IMP. Wherever decaffeination, caffeine is not usually chemically these in turn are transformed into xanthosine and synthesized. If desired, it may be synthesized from

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dimethylurea and malonic acid. The crude extract then lead to the formation of 7- of Escherichia coli catalyzes the removal of the methylxanthine.then; caffeine is formed in vitro Ribose moiety from 7-methyl xanthosine; and from the conversion of xanthosine [7].

Figure 2.Caffeine Biosynthesis 1.4. Effect of Caffeine on Health lowered birth weight and smaller head circumference when it is > 300 mg per day [6]. Although caffeine is commonly assimilated for the enhancement of alertness and the performance As for caffeine’s effect on children, one study improvement, its use should be avoided by assessed the physiological effects of caffeine on pregnant women, children, and persons with young children ages 7 to 9 years old. The study cardiovascular disease and anxiety disorders. For demonstrated that, in both boys and girls, caffeine instance, studies have demonstrated a link can produce a lower heart rate and higher blood between caffeine ingestion and risk elevation of pressure. Caffeine may also affect sleep patterns miscarriage. One study supporting these in teenagers. Because of caffeine’s adverse effects, determination states that the risk of miscarriage some people may choose to control and/or reduce is doubled when the ingesting > 300 mg per day their caffeine intake. Caffeine is most commonly of caffeine compared to women whose caffeine taken through coffee, and therefore, many intake is < 151 mg per day. Another study showed websites suggest switching to tea in order to limit that caffeine consumption is associated with daily caffeine intake[4].

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In addition to its most recognized stimulatory seeds are decaffeinated when they are still green. effect on the central nervous system, caffeine also However, many methods can remove caffeine affects several other tissues and organs, including from coffee, but all involve either soaking the the kidney, where it increases water and mineral green seeds in hot water (often called the "Swiss urinary excretion .Thus, the acute ingestion of water process" or steaming them, and then using caffeine in amounts increases the urinary solvents to dissolve caffeine-containing oils: excretion of calcium, magnesium, and sodium for Certain extracting solvents are including organic at least 3 h approximately after consumption. solvents such as dichloromethane (DCM); Water Renal conservation over 24 h after a caffeine load Extraction will be adopted as other polar my appear insufficient to offset the caffeine- molecules such as tannins which will also be induced calcium and magnesium losses[8]. extracted in the process; Supercritical Fluid CO2 and other recent methods of extracting caffeine Coffee is most well known as a beverage, it is also including High-Performance Liquid used as an ingredient in cooking. Wherever Chromatography(HPLC) and UV-Visible coffee's adverse effects are more common when spectrometer[9]. taken in excess. This is therefore the risks of coffee on health depend on its caffeine content[9]. 2. METHODS AND MATERIALS This study was performed in order to determine 2.1. Area of Study and Collection of samples Caffeine content in coffee growing in different The collection of coffee samples was done from Regions of Rwanda. The results showed the coffee region that contains the highest amount of the coffee grown in different regions of Rwanda (Figure 2). Those different coffee growing regions caffeine. Several species quantity of caffeine content varies widely depending on the types of are MUSASA Coffee (GAKENKE District located in the Northern Province, GASHONGA Coffee (RUSIZI which coffee is extracted such as coffee Arabica; coffee Robusta and Libelica [5]. which are located in the Western Province, MARABA Coffee (HUYE District) are located in 1.5. Decaffeination process Southern Province, HUMURE Coffee (GATSIBO

District) located in Eastern province and were Decaffeination may be defined as part of the found at NAEB. processing that coffee seeds undergo. Wherever,

Figure 3. Location map of the coffee-growing region of Rwanda.

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2.2. Determination of Caffeine content in Coffee to room temperature (25oC). Then after, 40 ml of Using UV-Visible spectrometer dichloromethane (CH2CL2) was poured into the coffee infusion and stirred for 10 min with a

2.2.1. Experimental Design magnetic stirrer at 1000rpm. Since caffeine is more soluble in dichloromethane (140 mg/ml) than it is in water (22 mg/ml), therefore, it readily dissolved in dichloromethane. The water phase and the dichloromethane phase were separated by a separatory funnel. The volume of dichloromethane was measured. Then, the dilution of the coffee was prepared by pipetting 10 ml into 200 ml volumetric flasks and diluting to 250 ml with distilled water.

The caffeine standards were used to identify the caffeine peak. Whereby, the peak area increased Figure 4. Quantification steps of caffeine content from the lowest standard to the highest and then in Coffee by UV-VIS Spectrometer he caffeine peak was used to determine caffeine present in the samples by plotting relation 2.2.2. Standard Preparation between absorbance and concentrations in the The characterization and determination of standard calibration curve. The UV-Visible caffeine content from different samples of coffee, spectrometer parameter was: wavelength at 650 were performed regarding to the solutions of nm[10]. standard caffeine in different solvents. The 2.2.4. Statistical analysis standard solutions were prepared as follow: 0.46 mg, 1.40 mg, 0.80 mg of pure caffeine was Caffeine content results were analyzed and one- immersed into 25 ml, 50 ml, 50 ml, and 40 ml of way ANOVA was used in comparison of the dichloromethane, distilled water, chloroform, and caffeine levels’ means according to the coffee ethyl acetate respectively, and stirred by a growing in different regions of Rwanda. The magnetic stirrer for 30 min. Then after, the standard deviation was also calculated. relationship between absorbance and wavelength of the solution was performed using a UV-Visible 3. RESULTS AND DISCUSSION spectrometer. Wherever, the procedures were The study on Quantitative Analysis of caffeine triplicated and finally, the molar absorption content from coffee growing in different regions of coefficient, transitional dipole moment, and Rwanda was conducted in laboratory. The results integrated absorption coefficient were calculated. were obtained by identification test, gravimetric

2.2.3. Samples preparation and analysis method, and ANOVA and discussed as below.

Firstly, 30 ml of distilled water was heated to 30°c 3.1. Results and 50 mg of coffee were put into the hot water Table 2 below presents the results of absorbance and the mixture was stirred for 30 min with a and concentrations of caffeine standard obtained magnetic stirrer at 1000rpm, the stirred solution via UV-Visible spectrometer for four standard was then filtered through a glass filter and cooled solutions. Table 2.Results (absorbance) and the concentrations of caffeine standard. No of standard solution Concentration in ppm absorbance Transmittance% 1 2.788 0.006 98.6 2 4.848 0.026 93.8 3 5.98 0.034 90.4 4 8.85 0.052 82.9

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2 have been used to establish a calibration curve represented by figure 5. Four different concentrations of caffeine standard 2.788 ppm to 8.85ppm as mentioned in the table 0.06 y = 0.0075x - 0.0125 0.05 R² = 0.9848

0.04

0.03

Absorbance Absorbance 0.02

0.01

0 0 2 4 6 8 10 Concentration ppm

Figure 5. Calibration Curve for caffeine Standard. From the calibration curve obtained, the regression equation was presented as follow: Correlation coefficient R2 Slope(m) y-Intercept Linear range in ppm

0.9848 0.0075 0.0125 2.78-8.85

The curve absorbance vs. concentration has The equation was: y=0.0075x-0.0125 (1) shown a good linear relationship. The curve was made in order to analyze the results using the where y=Absorbance ,x=Concentration in ppm equation to find the concentration of caffeine in unknown four different coffee samples. Table 2. Caffeine Concentration (ppm) in each coffee sample from four regions of Rwanda Regions Caffeine concentration in ppm Standard deviation Eastern province 3.2667 1.578 Western province 9.4 1.962 Northern province 3.8 1.270 Southern province 7.5333 0.8885

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Concentration ppmConcentration in 0 Eastern province Western province Northern province Southern province Coffee growing Regions

Figure 6.Comparison of caffeine content (ppm) in coffee from four regions of Rwanda caffeine concentration. The third coffee in caffeine concentration is the one grown in Northern From table 2, the highest caffeine concentration Province region presented by MUSASA Coffee was obtained in the coffee sample grown in the (Gakenke District) which contains 3.8±1.270 ppm. Western province region presented by The last caffeine concentration was obtained in GASHONGA Coffee (Rusizi district) with 9.4±1.962 coffee from Eastern province, presented by ppm. This was followed by coffee grown in the HUMURE Coffee (GATSIBO District) with a caffeine Southern province region presented by MARABA concentration of 3.2667±1.578ppm as presented Coffee which contains 7.5333±0.8885 ppm as in figure 7.

Concentration ppmConcentration in 2

0 Western province Southern province Northern province Eastern province

Coffee Growing Regions

Figure 7. Comparison of caffeine content (ppm) 3.2. Discussion The research showed positive results matched

with the question asked before, where the coffee This research has been conducted at UR-CST presenting four regions of Rwanda contain

Nyarugenge Campus Laboratories. This research different caffeine content. was entitled “Quantitative analysis of caffeine content in coffee from a different region of The results of our study support the previous Rwanda. work done by Placide where he analyzed the

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caffeine content from tea growing in different coffee (GAKENKE District) that presents Northern regions of Rwanda. Province, which gave caffeine content of 3.8±1.270 ppm. The least coffee in caffeine content was taken

The findings in figure 7 are in the same line with from HUMURE coffee (GATSIBO District) that the previous study reported by Placide where he presents the eastern province, with a caffeine found out that the tea with high caffeine content content of 3.2667±1.578ppm. was in Rubaya tea presenting the Western province, the second was the Mata tea presenting From the results, we generally conclude that the Southern province, the third tea was from Mulindi coffee-growing from four regions of Rwanda in the Northern province and the last one was contains different caffeine content and it was Sake tea that presented Eastern province. This ranged from 3.2667±1.578 ppm to 9.4±1.962ppm. study showed that Coffee growing in different The order of coffee in caffeine content from the regions was also found to have different caffeine highest was: GASHONGA Coffee > MARABA Coffee content. This difference may due to climate, > MUSASA Coffee > HUMURE Coffee. abundant rainfall, acidic soils, and altitude of the land above sea level. Based on the altitude which Further research should be done on this topic and try to collect more samples from each region in affects caffeine, Rwandan coffee planted on hillsides at high altitude (between 1,900 and 2,500 order to get efficient results. m) contains a high amount of caffeine content, this 5. ACKNOWLEDGMENTS can be caused by its good condition required for coffee growth which affect also the caffeine This research was conducted in the University of content. Those hills region receives abundant Rwanda –College of Science and Technology rainfall, are indicated by low temperature and laboratory, I a appreciate University for providing high humidity and acidic soils whose PH varies laboratory space and facilities. I also wish to between 4.5 to 5 that contribute to the coffee express my sincerest appreciation to my growth and high caffeine content in general supervisor Dr.NSHOGOZA Gilbert for the advice, [11].However, the coffee planted on well-drained support, and guidance he offered throughout this marshes at an altitude between 1,550 and 1,800 project period. m have low concentration of caffeine content REFERENCES comparing to the coffee from the hill region with higher altitudes like the Western part of our [1] A. Fodor, “Comparative studies about country. This may due to the high temperature, caffeine content in roasted ground coffee and low humidity, soil pH of 5 to 5.5, and low level of china black tea,” vol. VII, pp. 966–971, 2008. the rainfall that characterize the marshes regions like in the Eastern part of Rwanda where we found [2] A. Belšc and D. Horz, “Comparative study the coffee with low caffeine [11]. of polyphenols and caffeine in different coffee varieties affected by the degree of roasting ˇ 4. CONCLUSION movie,” vol. 129, pp. 991–1000, 2011, doi: 10.1016/j.foodchem.2011.05.059. The objective of this study was to quantify the caffeine content from coffee growing in different [3] A. T. Dado, Y. A. Asresahegn, and K. G. Regions of Rwanda. The results helped the Goroya, “Comparative study of caffeine content in consumers and researchers to identify the coffee beans and leaves of Coffea arabica using UV / Vis region that contains the highest amount of spectrophotometer,” vol. 14, no. 14, pp. 171–176, caffeine. The laboratory results showed that the 2019, doi: 10.5897/IJPS2019.4814. coffee presenting different regions of Rwanda contains different caffeine content. The highest [4] J. M. Chin, M. L. Merves, B. A. Goldberger, caffeine quantity was obtained in the coffee A. Sampson-cone, and E. J. Cone, “Caffeine Content sample grown at GASHONGA Coffee (RUSIZI of Brewed Teas,” vol. 32, no. October, pp. 702–704,

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