1. INTRODUCTION:

Caffeine is one of the most popular consumed pharmacologically active substance in the world due to its central nervous system stimulant activity and analgesic property¹.

Caffeine is present naturally in different amounts in the beans, leaves and fruits of about sixty herbs and trees, higher concentration of caffeine is present in the kola nut , cacao bean , mate and guarana berries²; moreover, overheated coffee beans (Coffeaarabica and Coffearobusta), and the leaf of tea (Cameliasiniensis) are the world's major sources of nutrient caffeine^3,4, Figure1.

Figure 1: Caffeine content in some drinks and food

Nowadays a lot of caffeinated drinks and juices present in the market like energy dinks, sport drinks and other beverages⁵.

Also many medications, dietary supplements and pain relieve drugs contain caffeine in different concentration⁶.

Caffeine is a purine alkaloid derived from methylxanthine which have heterocyclic ring similar to that of adenine in DNA and guanine in DNA and RNA⁷, Figure 2.

Figure 2:caffeineStructure

Rapid action of Caffeine is related to its permeabilitythrough the blood-brain barrier and adenosine receptors stimulation, resulting in antagonism to all receptor subtypes⁸.

Most peopleregulate their caffeine consumptionin according with the objective and subjective effects of the methylxanthine⁹.

However, individual response to caffeine varies greatly from person to person due to the variation in metabolism, age, sex, hormones, clearance, weight, genes, medicine intake and smoking behavior, therefore the amount of caffeine required to achieve the desired effect also varies greatly¹⁰.

In this paper we have estimated the quantity of caffeine in different commercial kinds of coffeepresent in the market.

2. MATERIAL AND METHODS:

2.1 Coffee brands:

Different commercial kinds of coffee have been collected from the market, Figure3.

Figure3: Coffee brands used in the study

2.2 Isolation of caffeine from coffee:

Caffeine has been extracted by boiling 50g of coffee powder (for each type) with 250ml of distilled water on heater with continuedstirring for 15min, the extract then filtered through filter paper while still hot and a solution of basic lead acetate was added to the filtrate until no more precipitate is formed, then after centrifugation, diluted sulphuric acid was added to the hot supernatant until precipitation ceases. Then 1g of charcoal is added to the filtrate, which is then evaporated over a low Bunsen flame with frequent stirring until a volume of 100 ml is reached, the solution is then filtered hot many times until a clear filtrate is obtained.

The filtrate then left for cooling and partitioned with chloroform, then the chloroform layer was evaporated on steam bath in the hood the residue Scraped out, transferred to a small beaker and dissolved in the smallest quantity of hot 60°C ethanol for crystallization¹¹.

2.3. Identification of caffeine crystals:

2.3. 1Qualtitative Analysis:

A) Specific test: The murexide test¹²

Small amount of potassium chlorate was added to a few crystals of caffeine in porcelain dish and then 2drops of concentrated Hcl were added, the mixture was evaporated to dryness, and then expose to ammonia vapor.

B) T.L.C

Three solvent systems have been used for caffeine identification using TLC Gf₂₄₅ in comparison with caffeine standard and UV₂₄₅ detection.

S1: Acetone : Water : Ammonia ( 9:0.7:0.3(¹³

S2: Methanol : NH4OH ( 100:1(¹⁴

S3: Isopropanol : Acetic Acid : H20 (6:2:2(¹⁵

C) FTIR spectra were done by Shimadzu at the college of pharmacy /Mustansiryiah University.

2.3.2 Quantitative Analysis:

This is done simply by weighing the crystals of the caffeine.

3. RESULTS AND DISCUSSIONS:

Crystals of caffeine were obtainedand weighted, the weight and the percent of caffeine has been calculated and all the results were tabled in table 1.

Table (1): Weight of caffeine extracted

Coffeebrand	Country	Coffee weight	Weight of caffeine extracted	%of caffeine
Almawakeb	India	45 gm	0.54 gm	1.2%
MasCafe	India	50 gm	0.576 gm	1.152%
Brazilian	India	50 gm	0.081 gm	0.162%
Coffee Break	jordan	50 gm	0.095 gm	0.19%
Cafe Pele	Brazil	50 gm	0.05 gm	0.1%

The coffee with the highest amount of caffeine was (ALMAWAKEB) Coffee brand followed by (MasCafe), (COFFEE BREAK), (BARAZILIAN) and (CAFE PELE) sequentially. Coffee with the lowest amount of caffeine was (CAFE PELE) Coffee brand.

Purines unlike other alkaloids do not give positive results with general tests of alkaloids; therefore murexide test is used in its identification. all the crystals give a pink color which is considered a positive result for purine alkaloids.

and all the crystals give the same Rf value of caffeine standard in the three mobile phases used in the study as shown in (Figure 4) and (table 2)

Table 2: Rf value of caffeine crystals compared with caffeine standard

Rf value						Mobile phase
St	1	2	3	4	5	Mobile phase
0.78	0.82	0.78	0.79	0.81	0.8	S1
0.68	0.67	0.66	0.7	0.7	0.7	S2
0.44	0.46	0.46	0.44	0.42	0.42	S3

The IR spectra of the isolated caffeine as compared with standard are shown below in figure 5 and the absorption data of functional groups are presented in table 3, the comparable value between the functional group absorption of the isolated caffeine with that of standard confirms the correct structure of the isolated caffeine.

Figure 4: TLC of caffeine crystal extracted from coffee

1:MasCafe, 2:CAFE PELE, 3:COFFEE BREAK, 4:ALMAWAKEB, 5: BRAZILIAN, X:Caffeine standard

Functional group	Wave length (cm^-1) of caffeine
Functional group	standard	Mas cafe	Café pele	Coffee break	brazilian	almawakeb
Ѵ C-H	3109	3110	3110	3114	3116	3117
Ѵ C=O	1695	1693	1694	1696	1704	1697
Ѵ C-N	1024,1188,1238	1023,1187,1236	1023,1186,1236	1020,1187,1236	1023,1236	1020,1187,1237
Ѵ C=N	1598	1598	1598	1548	1550	1549
CH₃bending	1325	1325	1325	1335	1316	1336
C-H deformation	758	758	758	758	759	759

Figure5: IR charts of standard and isolated caffeine

The difference in caffeine percent between different brands may be related to thedifference in the coffee beans source from different country growing under different environmental conditions¹⁶ which consider the major sources of secondary metabolites variation in plant species, Those includeregional adaptation or acclimation, weather differences due to geographical differences¹⁷, also the time of collection¹⁸and the process of bean manufacture also affect the percent of caffeine¹⁹.

CONCLUSION AND RECOMMENDATION:

The major source for caffeine is coffee and different coffee branch contain different percent of caffeine therefore caffeine intake must be adjusted to reach the desired effects in each person due to individual differences.

So, each person should take care and avoid consuming a large quantity of coffee and not exceed the recommended quantity, especially if they have chronic diseases or take some medication.

ACKNOWLEDGMENT:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad-Iraq for its support in the present work.

REFERENCES:

1. Chowdhury SR, MalequeM, ShihanMH. Development and Validation of a Simple RP-HPLC Method for Determination of Caffeine in Pharmaceutical Dosage Forms. Asian J. Pharm. Ana. 2012 Jan-Mar;2(1):1-4.DOI:10.5958/2231-5675.2019.00039.5

2. Shanthakumar J., Tamilselvan T., Arunagiriet.al. Role of caffeine intake in lithium treated methylphenidate induced oxidative stress in an animal model of mania. Asian J. Pharm. Res. 2013Oct. - Dec;3(4):166-171. DOI: Not Available

3. Deepth BVP and MuthuprasannaP. Effect of Surfactant on Extraction of Caffeine from Coffee Powder. Research J. Pharmacognosy and Phytochemistry 2012; 4(5): 271-276.DOI: Not Available.

4. SetyawanEI, SetyowatiEP, Abdul Rohman et.al. Simultaneous Determination of EpigallocatechinGallate, Catechin, and Caffeine from Green Tea Leaves (Camellia sinensis L) Extract by RP-HPLC. Research J. Pharm. and Tech 2020; 13(3):1489-1494. http://doi.org:10.5958/0974-360X.2020.00271.1

5. PatilP A, Suhas Mane, AtulWakse, RavikumarSawant. Evaluation of tea waste for Nitrogen, Phosphorous, Potassium (NPK) as Organic Fertilizer. Asian J. Res. Pharm. Sci. 2018; 8(4):217-218. http://doi.org:10.5958/2231- 5659.2018.00036.X.

6. RoyA. andDasB.Effects of Caffeine on Health: A Review. Research J. Pharm. and Tech. 2015 Sep; 8(9):1312-1319.http://doi.org:10.5958/0974-360X.2015.00237.1

7. Lella R., Mathew EM andMoorkothS. Development and Validation of a LC-MS2 Method for the Simultaneous Quantification of Caffeine and Adenosine from DBS. Research J. Pharm. and Tech. 2019; 12(12): 5878-5882. http://Doi.org:10.5958/0974-360X.2019.01019.9

8. KumarSB. Role of Caffeine in Dementia, Alzheimer’s, Parkinsonism, Bipolar Mood Disorder. Research J. Pharm. and Tech. 2015 Nov;8(11):1582-1587.http://DOI.org:10.5958/0974-360X.2015.00282.6

9. El-Nabarawi MA, Teaima MH and Hamid et.al. Formulation, Evaluation and Antioxidant activity of Caffeine Fast Melt Tablets. Research J. Pharm. and Tech 2018;11(7):3131-3138.http://DOI.org:10.5958/0974-360X.2018.00575.9

10. M. Muthukkaruppan, A. Nithya, P. Parthiban. Caffeine Estimation in Darjeeling District Tea Samples. Research J. Pharm. and Tech 2018; 11(5):1981-1983.http://DOI.org:10.5958/0974-360X.2018.00368.2

11. Olechno, E., Pu´scionJakubik, A., Zujko, M.E.,Socha, K.Influence of Various Factors on Caffeine Content in Coffee Brews. Foods 2021 May; 10(6):1208. https://doi.org/10.3390/foods10061208

12. Hamad M.N. and Abdul-Hussain D. A. Gravimetric Estimation of Caffeine in Different Commercial Kinds of Tea Found in the Iraqi Market. Iraqi J Pharm Sci. 2010 Mar; 19(2): 48-53.https://doi.org/10.31351/vol19iss2pp48-53

13. Blakseley J., Wood D., Howse C. and Spencer-Peet J. A simplified thin-layer chromatography system for the detection of commonly abused basic drugs. Ann. Clin. Biochem. 1987 Sep; 24(5):508-510.https://doi.org/10.1177/000456328702400514

14. El-Bagary R.I., Mohammed N.G. and Nasr H.A. Two Chromatographic Methods for the Determination of Some Antimigraine Drugs. Anal Chem Insights. 2012 May; 7: 13–21.https://doi.org/10.4137/ACI.S8864

15. Allison RT. and Garratt NJ. Solvent systems for thin layer chromatography of biological dyes. Med. Lab. Sci. 1989 Apr; 46(2):113-9.PMID: 2593771.

16. Fox GP., Wu A., Liang Y and Force L. Variation in caffeine concentration in single coffee beans. Journal of Agricultural and Food Chemistry 2013 Nov 13; 61(45):10772-8. http://DOI.org:10.1021/jf4011388

17. Hongjie M., Wei X., Franklin SB. and Wu H. Geographical variation and the role of climate in leaf trait of a relic tree species across its distribution in China. Plant Biology 2017 Jul; 19(4):552-561. http://DOI.org:10.1111/plb.12564

18. Chen X., Ma Z. and Kitts DD. Effects of processing method and age of leaves on phytochemical profiles and bioactivity of coffee leaves. Food Biochemistry 2018 May; 249:143-153.http://DOI.org:10.1016/j.foodchem.2017.12.073

19. Bastian F. and Syarifuddin A. Effect of decaffeination and re-fermentation on level of caffeine, chlorogenic acid and total acid in green bean robusta coffee. IOP Conference series earth and environmental science 2021; 807(2):022069.https://doi.org/10.1088/1755-1315/807/2/022069

20. Cordoba N., Pataquiva L. and Ruiz RY. Effect of grinding, extraction time and type of coffee on the physicochemical and flavour characteristics of cold brew coffee. Sci. Rep. 2019 Jun 11; 9(1):844.http://DOI.org:10.1038/s41598-019-44886-w

Received on 31.07.2022 Modified on 14.10.2022

Research J. Pharm. and Tech 2023; 16(7):3358-3362.

DOI: 10.52711/0974-360X.2023.00555