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The Comparative Qualitative and Quantative Analysis on Green and Black Tea (Camellia sinensis) Extracted by Three Solvents Studied in Sudan

The Comparative Qualitative and Quantative Analysis on Green and Black Tea (Camellia sinensis) Extracted by Three Solvents Studied in Sudan

By

Murwan K. Sabah EL-Kheir 1

1. Department of Biochemistry, School of Biotechnology, Faculty of Science and

Technology, El Neelain University, P.O.Box 12702, Khartoum, Sudan

Email address; murwansabahelkhier@yahoo.com

Abstract: The aim of this study is to investigate the phytochemicals screen of Green and Black Tea extracted by water, ethanol and petroleum ether. The results revealed that the presence of alkaloid, tannin, steroid, flavonoid and carbohydrate in Green and Black Tea extracted water ethanol and petroleum ether. In addition, Green and Black Tea extracted by water contained saponins, but Green and Black Tea extracted by ethanol and petroleum ether do not contained saponins.Green and Black Tea extracted by ethanol and petroleum ether contained cardenolide, but Green and Black Tea extracted by water do not contained cardenolide and finally Green and Black Tea extracted by water ethanol and petroleum ether do not contain anthraquiaone and coumarin. The alkaloid content of Green Tea extracted by water, ethanol and petroleum ether is 5.4, 4.3 and 4.6 Mg/g, respectively. While alkaloid content of Black Tea extracted by water, ethanol and petroleum ether is 5.4, 4.3, and 4.Mg/g, respectively. The tannin contents of Green Tea extracted by water, ethanol and petroleum ether are 1.2, 1.8 and 1.5 %, respectively. While tannin contents of Black Tea extracted by water, ethanol and petroleum ether are 1.2, 0.5, 1 %, respectively.

Keywords: Tea, Alkaloid, Tannin and Qualitative.

1.0 Introduction

Green and Black Tea is made up from Camellia sinensis, which is characterized as a perennial evergreen shrub. Tea varieties reflected the growing region (Ceylon and Assam), districted (Darjeeling), form (pekoe and gunpowder) and processing method (green, black and oolong). India and Srilanka is major producers of green tea. The Turkish trader’s reportedly introduced to Western culture in 6th Century (1) .It has many uses for its mild stimulant and medicinal properties (prevent cancer (2), prostate cancer (3), asthma (4), dental cavity, diabetes (5), heart attack (6), high cholesterol (7), improve fertility (3), memory enhancement, mental performance and menopausal symptoms). The green tea is prepared by exposing the gathered leaves to air until superfluous moisture is eliminated, leaves roasted over a brick wood fire and continually stirred until leaves become moist and flaccid, after which leaves pass to rolling table and rolled into balls and subjected to pressure which twist them and gets rid of the moisture, then leaves shaking out on flat trays, again leaves roasted over a slow and steady charcoal fire and kept leaves in rapid motion for an hour  to hour and half., till leaves assume a dullish green colour after that the leaves winnowed, screened, and graded into different types. While black tea, gathered leaves are exposed to air for long period, gathered up, tossed until soft and flaccid, further exposure, roasted in an iron pan for about five minutes and after rolling and pressing the leaves are shaken out, exposed to the outer air for some hours, re-roasted for three or four minutes and recoiled, spread out in baskets and exposed to heat of charcoal fire for five or six minutes and then rolled for third time and again heat and finally dried in baskets over charcoal fire from which leaves become black in colour. Steroid compounds are containing the perhydrocyclopentano-phenanthrene skeleton and usually occur in glycosidal combination with sugar (8). Alkaloids are group of natural products (atropine, quinine, morphine …etc) which are widely used to treat Malaria and Cancer diseases (8). Cardenolides are act on the heart direct or indirect mechanism to enhance the force and velocity of the contraction, therefore it is known as cardiotonic (8). Carbohydrates are the most abundant organic constituents of plant, serve as the major source of chemical energy (sugar and starch), as well as important constituents of supporting tissue (cellulose in wood, cotton and flax) (8). Anthraquinones are oxygenated derivative of pharmacological importance that is used as laxatives or cathartics, anti-inflammatory, antibacterial, antifungal and also as natural dyes (8). The flavonoid, constitute one of most diverse and widespread group of natural products, found in fruit, vegetables, nuts, seeds, stem, flowers, as well as tea, and are one of the most important constituents of the human diet(8). Coumarins are derivative of benz-? pyrone, or lactones of o-hydroxcinnamic acid such as coumarin, umbelliferonee, aesculetin, daphnetin, fraxetin and scopoletin, they are found in both state in free or combination with glycosidic., not all members of the group are phenolic (8).The tannin compounds are phenolic polymers precipitate protein from aqueous solution and it is reduce or inhibit enzyme activity (9).

Objectives of this study are Phytochemicals screening of the green and black tea, which included:(1) Qualitative analysis of Alkaloid, Anthraquiaones, Cardenolides, Tannin, Saponins, Coumarin, Flavonoid, Steroid and Carbohydrates in green and black tea extracted by water, ethanol and petroleum ether in green and black tea extracted by water, ethanol and petroleum ether. (2) Quantative analysis of Alkaloid and Tannin content

2.0 Materials and methods

2.1 Sample preparation: The dried green and black teas were collected from local market of Khartoum State, Sudan., the dried teas (green and black) were freed from foreign materials and ground into fine powder to pass 0.4 mm mesh screen. The prepared samples were kept in tight containers and stored at room temperature until analysis. Preparation of e aqueous extraction of tea (green and black) by using water, ethanol and petroleum ether is carried out according to method described by (10).

2.2 Qualitative analysis: Alkaloid, Saponins, Tannin, Steroid, Flavonoids, Comrine, Cardiac and Carbohydrates were identified according to method described by (10).

2.2.1 Alkaloid: Two gram of well ground samples was extracted with 10 ml of 1%HCL for 30 minutes in water bath. The suspension was filtrated through cotton whool into tube and the filtrate solution was divided into two portions A and B. Then saturated solution of sodium bicarbonate was added to the filtrate solution (B) till pH become 8 -9 and the solution was mixed with 3 ml chloroform, then upper layer was removed by pipette and treated with acetic acid till pH become 5 and five drops of dragendroff,s regent were added. If the precipitate is form. This indicated the presence of quaternary alkaloid. The lower layer (chloroform) was treated with 3 ml of 1%HCL which separated the lower layer into two layers. Then upper layer was pipette and treated with dragendroff,s regent, this indicated to presence of tertiary alkaloid.

2.2.2 Anthraquinones: 0.5 gram of well ground sample was extracted in boiling water for two minutes with 5 ml 0.5N KOH and 0.5 ml H2O. Suspension was cooled and filtrate by using glass wool and six drops of acetic acid was added, then the solution was mixed with 5 ml benzene, The upper layer was separated and pipette into test tube followed by adding of 2 ml 0.0N KOH. If the solution gives red colour. This indicated the presence of Anthraquinones.

2.2.3 Cardenolides: 3 gram of well ground sample was extracted 2 ml of distil water in boiling water bath for 30 minutes, then allow the solution to cool and filtrate by using glass wool into test tube. 10 drops of lead acetate was added and then filtrate to tube (A) and (B). Four drops of Keddle,s reagent was added The solution (B) was mixed with 2 ml chloroform, and 4 drops of Keddle,s reagent was added to lower layer. If the solution gives a violet colour. This indicated the presence of Cardenolides.

2.2.4 Tannin: 3 gram of well ground sample was extracted with 1 ml distil water in boiling water bath for 5 minutes, then filtrate the solution by using filter paper and allow a solution to cool . The filtration solution was divided into two portions A and B. 5 ml of 2% NaCL was added to portion A. Suspended solution was filtrated by using filter paper, followed by adding 5 ml 1% gelatin solution. Precipitate indicated the presence of Tannin. 1 ml of 1% ferric chloride was added to portion B. The green blue colour indicated the presence of Tannin.

2.2.5 Saponins: 3 gram of well ground sample was extracted with 5 ml distil water in boiling water bath for 6 minutes. Then filtrate the solution by using glass wool. 1 ml of sample extracted was shaken for 5 minutes and finally the persistent and voluminous froth is formed.

2.2.6 Coumarin: 3 gram of well ground sample was extracted with 5 ml distil water in boiling water bath for 6 minutes, then filtrate the solution by using filter paper and allow a solution to cool. Then the sample extracted shows a blue fluorescence, but it changed into greenish - blue on addition of few drops of ammonia. This indicated to presence of coumarin.

2.2.7 Flavonoid: 3 gram of well ground sample was extracted 2 ml of distil water in boiling water bath for 30 minutes, then allow the solution to cool and filtrate by using glass wool into test tube. 10 drops of lead acetate was added to filtration. The yellowish precipitate is formed. This indicated to presence of Flavonoid.

2.2.8 Steroid: 3 gram of well ground sample was extracted with 5 ml ethanol with 1% acetic acid in boiling water bath for 5 minutes, then filtrate the solution by using filter paper and allow a solution to cool. The filtration solution was divided into two portions A and B. Few drops of SbCL3 was added to portion A and few drops of Marquis’s reagent was added to portion B. red colour develop in portion A and violet to red colour develop in portion B. This indicated to presence of Steroid.

2.2.9 Carbohydrates: 3 gram of well ground sample was extracted with 5 ml distil water in boiling water bath for 6 minutes. Then filtration the solution by using glass wool. Few drops of Molish, s reagent were added to filtration solution. Then violet colour appeared, this indicated to presence of Carbohydrates.

2.3 Quantative analysis:

2.3.1 Alkaloid content: It is determined according to method described by (8).

Evaporated combined extract on steam bath with air current to about 10 ml add measured excess 0.2N H2SO4 and continue evaporation to remove the solvent, cool and add methyl. Then the excess acid (H2SO4) was titrated with 0.02 N NaOH.

V1 X M1 = V2 X M2 (1)

Mg/g = V2 X D.F. X M.WT (2)

103 X S

Where: V1 = Volume of 0.20.2N H2SO4, M1 = Concentration of H2SO4 (0.2N), V2 = Volume of NaOH, M2 = Concentration of NaOH (0.02 N), D.F. = Dilution factor, M.WT = Molecular weight of NaOH, S= Weight of sample and 103 = conversion factor from volume to weight.

2.3.2 Tannin content: It is determined according to method described by (11).

0.2 gram of extracted sample was placed in 50 ml of conical flask and 10 ml of 1% HCL was added. The contents of flask were shaken for 20 minutes, and then contents of the flask were centrifuged at 3000 rpm for 5 minutes. Then contents of flask were incubated at 30 oC for 20 minutes. The spectrophotometer was read at 500 nm and then scale read zero with the blank solution. Finally, readings were taken three times and averaged.

1. C.E. (%) =    C X 100 (1)

10 X S

Where: C.E. = Catechin equivalent, C = Concentration corresponding to optical density, 10 = Volume of extract (ml) and S = Weight of sample.

3.0 Results and Discussion

3.1 Qualitative analysis: The qualitative test of green and black tea that extracted by water, ethanol and petroleum ether for the alkaloid, anthraquiaones, saponins, tannin, cardenolides, steroid, flavonoids, coumrine, and carbohydrates were shown in Table (1). The findings revealed that the alkaloid, tannin, steroid, flavanoid in three extractions (water, ethanol and petroleum ether) of green and black tea are available. The cardenolide in two extractions (ethanol and petroleum ether) of green and black tea are presence but the cardenolide in water extraction is not found in green and black tea. These results indicated that cardenolide is not dissolved in water but dissolved in ethanol and petroleum ether. In addition to that the anthraquiaones and coumarin three extractions (water, ethanol and petroleum ether) of green and black tea are not available. The saponin in green and black tea extracted by water is present, but saponin in green and black tea extracted by ethanol and petroleum ether is not present  These findings revealed that green and black tea do not contain anthraquiaones and coumarin.These results are supported by those results given by (12) ..

3.2 Quantative analysis: Table (2) indicated that the alkaloid content in green tea in water, ethanol and petroleum extracted is 5.4, 4.3 and 4.6%, respectively. While the alkaloid content in black tea in water, ethanol and petroleum extracted is 6.4, 4.5 and 5.2 %, respectively. The results revealed that alkaloid in both green and black tea in water extracted is higher than in ethanol and petroleum ether extracted, but there is no significant difference in three solvents extracted for green tea at (P ? 0.05).This indicated that extraction of alkaloid is not effected by types of solvents. In addition to that the results indicated that tannin content in green and black tea for water extracted is similar, tannin content in green tea for ethanol extracted is higher than in black tea. The results revealed there significant difference at (P ? 0.05). The variation in alkaloid and tannin content is attributed to type of solvent, type of tea and process of tea.

Conclusion and Recommendation:

1. We concluded that the green and black tea contain alkaloid, tannin, steroid, flavonoid, cardenolides, saponins and carbohydrate. In addition amount of alkaloid and tannin are varied according to type of solvent used.  Our recommendation, the farther study in green and black tea should be done particularly antimicrobial study.                 

References

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(2) Chiu, A, E, Chan M.C.and Kern D.G. (2005). Double –blinded, placebo- controlled trial of green tea extracts in the clinical and histological appearance of photo aging skin.Dermatol Slurg. 31(7 pt 2):855 – 860.

(3) Chow H.H., Hakim, I.A., Vining D.R. (2005).Effects of dosing condition on the oral bioavailability of green tea catechins after single=dose administration of polyphenols in healthy individuals. Clin. Cancer Res. 11(12):4627-4633.

(4) Laurie S.A., Miller V.A., (2005). Study of green tea extract in patients with advanced lung cancer. Cancer Chemother Pharmacol. 55(1):33 -38.

(5) Fukino Y., Shimbo M. and Aoki N. (2005). Randomized controlled trial for an effect of green tea consumption on insulin resistance and inflammation markers.J.Nutr.Situation I (Tokyo) 51(5):335 -342.

(6) Riemersma R.A., Rice C.A., Tyrell R.M., Clifford M.C.and Lean M.E.(2001).Tea flavonoids and Cardiovascular Health. QJM.94 (5):277 -282.

(7) Maron D.J., Lu G.P and Cai  N.S (2003). Cholesterol – lowering effect of the aflavins- Enriched tea extract: a randomized controlled trial. Med, fl1/4 44 MJ.163 (12):1448 – 1453.

(8) AOAC. 1990. Association of Official Analytical Chemists. Official Methods of Analysis. 15th edition. Washington.

(9) Goldstein, J.L and T. Swain 1963. Change in tannin in ripening fruit. Phyto chemistry (2),371

(10) FAO (1991). Guide to Specification: general notices, general analytical techniques, identification tests, test solution and other reference materials. FAO Food and Nutrition paper 5 Rev.2 Rome.

(10) Price M.L. and Butler L.G. (1987).A critical evaluation of the Vanillin

1. Reactions as an assay for tannin in sorghum rain. J. Agric. Food chem. 26

(5): 1214-1218.

(12) Balbaa S.I., Hilal S.H. and Zaki A.Y. (1981). Medicinal Plant Constituents. 3rd

Ed., General Organization for University and Schoolbooks. Cairo.

Table (1): Chemical constituents that available (+ ve) and non-available (- ve) in green and black tea.

Solvent extraction /

Compounds

Water extraction

Ethanol extraction

Petroleum ether extraction

Green tea

Black tea

Green tea

Black tea

Green tea

Black tea

1/Alkaloid

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

2/Anthraquinones

(-) ve

(-) ve

(-) ve

(-) ve

(-) ve

(-) ve

3/Cardenolides

(-) ve

(-) ve

(+) ve

(+) ve

(+) ve

(+) ve

4/Tannin

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

5/Saponins

(+) ve

(+) ve

(-) ve

(-) ve

(-) ve

(-) ve

6/Coumarin

(-) ve

(-) ve

(-) ve

(-) ve

(-) ve

(-) ve

7/Flavonoids

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

8/Steroid

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

9/Carbohydrates

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

(+) ve

Table (2): Alkaloid and tannin content in extracted – tea solvent (water, ethanol and petroleum ether of green and black type.

Type of solvent

Water extraction

Ethanol extraction

Petroleum ether extraction

Type of tea

Green tea

Black tea

Green tea

Black tea

Green tea

Black tea

Alkaloid Mg/g

5.4a±1

6.4 a ±0.5

4.3 a ±0.3

4.5 a ±1.2

4.6 a ±1

5.2 a ±1.4

Tannin %

1.2 a ±0.5

1.2 a ±1

1.8 a ±2

0.5 b ±0.01

1.5 a ±1

1.0 a ±0.8

* Each value is average of three replicates expressed on dry weight basis.

About the Author

Murwan K. Sabah ELKheir 1
1. Department of Biochemistry, School of Biotechnology, Faculty of Science and Technology, El Neelain University, P.O.Box 12702, Khartoum, Sudan
Email address; murwansabahelkhier@yahoo.com

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