ISSN: 2454-1362, Food Value ... · 3. Add 1ml of distilled water to the test tube to make up the...

Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-10, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

Imperial Journal of Interdisciplinary Research (IJIR) 468

Food Value Analysis and Antioxidant Properties of Various Extracts of Rheum emodii

M. Amin Mir, Suraya Manzoor, Rabia Nazir, Rahilla Farooq,

Bilal Ahmad Dept. of Chemistry and Botany

Uttaranchal College of Science and Technology, Dehradun (India) Abstract: Rheum emodii plant have been analysed for the estimation of crude fibre, protein, ascorbic acid glucose content and the antioxidant potential. As per the results the plant shows a good concentration of all the metabolites. The plant was extracted using DCM, Methanol and Water as a solvent. The concentration of metabolites (Protein, Fibre, Ascorbic acid) shows increase in concentration as with the increase in the polarity of the solvent except glucose which shows highest concentration in DCM extract followed by water extract. Keywords: Protein content, Ascorbic Acid, Crude Fibre, Glucose, Rheum emodii. Introduction Medicinal plants are the plants used for centuries as remedies for human diseases because they contain components of therapeutic value. About 80% of world population relies on the use of traditional medicine which is predominantly based on plant material. The scientific studies available on a good number of medicinal plants indicate that promising phytochemicals can be developed for human health problems including cancer, diabetes and infectious diseases. Therefore the need to develop efficient, safe and inexpensive drugs from plant sources is of great importance. Most species are used only in folk medicinal, traditional system of formal medicinal using relatively few (e.g. 500-600 commonly in traditionally Chinese Medicine). Around 100 plant species have contributed significantly to modern drugs. The use of medicinal plants is increasing worldwide, related to the persistence and something expansion of traditional medicine and a growing interest in herbal treatment. According to Hina Rehman, Wajeeha Begum, Farzana Anjum, Humyra Tabasum (1) Rheum emodii or Himalayan rhubarb is a perennial herb belongs to family Polygonaceae. Rhizome of Rheum posses’ antibacterial, cytotoxic and antioxidant efficacies as mentioned by Richa K. Gupta, Gan B. Bajracharya, Ram Narayan Jha (2). The Study assessed by Amandeep Kaur, Sunil Kumar, Ramica Sharma (3) mentioned that R.

emodii have an excellent antioxidant and antiulcer potential. Sabreena Rashid, Zahoor A Kaloo, Seema Singh & Iram Bashir (4) Genus Rheum is medicinally important as it has hepatoprotective, spasmolytic, anti-cholesterolaemic, antitumor, antiseptic, antifungal, anti-microbial, anti-Parkinsons, anti-proliferative, immuno-enhancing, antiviral and antioxidant properties. R. emodii possess anticancer metabolites that can be isolated and used as precursors in development of anticancer drugs shown by V Rajkumar, Gunjan Guha, R. Ashok Kumar (5). Material and Methods Study area and plant collection The Rheum emodii rhizome was collected from the Anantnag region of Kashmir (J&K). The plant was shade dried and powdered in to mixture and the analysis of “Rheum emodii” was carried out at UTTARANCHAL COLLEGE OF SCIENCE AND TECHNOLOGY Dehradun Antioxidant Activity Determination DPPH Method DPPH Scavenging activity was measured by the spectrophotometric method. A stock solution of DPPH (1.5 mg/ml in methanol) was prepared such that 75 µl of it in 3 ml of methanol. Decrease in the absorbance in presence of sample extract at different concentration (10-125 µg/ml ) was noted after 15 min. IC50 was calculated from % inhibition. Protocol for DPPH Free Radical Scavenging Activity Preparation of stock solution of the sample:- 100 mg of extract was dissolved in 100 ml of methanol to get 1000 µg/ml solution. (1) Dilution of test solution: 10, 20, 30, 40,

50,60,70,80,90,100 µg/ml solution of test were prepared from stock solution.

(2) Preparation of DPPH solution: 15 mg for DPPH was dissolved in 10 ml of methanol. The resulting solution was covered with aluminium foil to protect from light.

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(3) Estimation of DPPH scavenging activity: 75 µl of DPPH solution was taken and the final volume was adjusted to 3 ml with methanol, absorbance was taken immediately at 517 nm for control reading. 75 µl of DPPH and 100 µl of the test sample of different concentration were put in a series of volumetric flasks and final volume was adjusted to 3 ml with methanol. Absorbance at zero time was taken in UV-Visible at 517 nm for each concentration. Final decrease in absorbance of DPPH with sample of different concentration was measured after 15 minute at 517 nm.

Percentage inhibitions of DPPH radical by test compound were determined by the following formula. % Reduction = Control absorbance – Test absorbance/ Control absorbance x 100 Calculation of IC50 value using graphical method. Estimation of Ascorbic Acid Sample Preparation Five grams of sample were homogenized with 25 ml of metaphosphoric acid acetic acid solution, and it was quantitatively transferred into a 50 ml volumetric flask and shaken gently to homogenized solution. Then it was dilute up to the mark by the metaphosphoric acid acetic acid solution. The obtained solution is filtered and centrifuged at 4000 rpm. Spectrophotometric determination (Perkin Elmer spectrophotometer lambda) of vitamin C content in nine different samples of plant extract Estimation of Vitamin C Procedure:- 0.23 ml of 3% bromine water were added into 4 ml of centrifuged sample solution to oxidize the ascorbic acid to dehydro Ascorbic acid and after that 0.13ml of 10% thiourea to remove the excess of bromine. Then 1 ml of 2, 4-DNPH solution was added to form osazone. All standards, samples and blank solution were kept at 37% temperature for 3 hours in a thermostatic bath. After it all were cooled in ice bath for 30 minutes and treated with 5ml chilled 85% H2SO4 with constant stirring. As a result, a coloured solution’s absorbance was taken at 521 nm. Procedure for Determination of Glucose Principle: - In hot acidic medium, glucose is dehydrated to hydroxyl methyl. This forms a green coloured product with phenol and had a absorption maximum at 490nm. Procedure 1. Weigh out 100mg of the sample in a boiling

tube.

2. Hydrolyze by keeping it in a boiling water bath for three hours with 5ml of 2.5 NaCl and cool it to room temperature.

3. Neutralize it with solid sodium carbonate until the effervescence ceases.

4. Make up the volume to 100 ml and centrifuge. 5. Pipette out 0.2, 0.4, 0.6, 0.8, and 1ml of the

working standards into a series of test tube. 6. Pipette out 0.1 and 0.2 ml of the sample

solution in two separate test tubes.make up the volume in each test tube to 1ml with H2O.

7. Set a blank with 1ml of H2O. 8. Add 5ml of 96% sulphuric acid to each test

tube and shake well. 9. Add 1ml of phenol solution to each test tube. 10. After 10 min shake the contents in the test tube

and place in a water bath at 25-300C for 30 minutes.

11. Read the colour at 490 nm. 12. Calculate the amount of total carbohydrates

present in the standard solution using the standard graph.

Procedure for Estimation of Amino-acid by Ninhydrin method The amino acids are colourless ionic compounds the forms the basic building blocks of proteins. Apart from being bound as proteins, amino acids also exist in the free form in many tissues and are known as free amino acid. They are mostly water soluble in nature. Very often in plants during disease conditions the free amino acid composition exhibits a change hence, the measurement of the total free amino acids gives the physiological and health states of the plants. Principle: - Ninhydrin, a powerful oxidizing agent, decarboxylates the alpha amino acids and yields an intensity coloured bluish purple product which is calorimetrically measured at 570nm. Reagent Required 1. Ninhydrin:- dissolve 0.8gm stannous chloride

(SnCl2.2H2O) in 500ml of 0.2M citrate buffer(pH 5.0) add this solution to 20gm of ninhydrin in 500ml of methyl cellosolve (2methoxy ethanol)

2. 0.2M Citrate Buffer pH5.0 Solution A: 0.2M citric acid. Solution B: 0.2M sodium citrate mix 20.5ml of solution A with 29.5ml of solution B and check pH.

3. Dilute Solvent: mix equal volume of water and n-propanol and use.

4. Standard: dissolve 50mg leucine in 50ml of distilled water in a volumetric flask. Take 10ml of this stock standard and dilute to 100ml in another flask for working standard solution.





Procedure 1. Pipette out 0.2, 0.4, 0.6, 0.8, 1.0 ml of standard

amino acid solution to the resp. labeled test tubes.

2. Add distilled water in all the test tubes and make up the volume to 1ml.

3. Add 1ml of distilled water to the test tube to make up the volume to 1ml.

4. Now add 1ml of ninhydrin reagent to all the test tube including the blank test tube and unknown test tube.

5. Mix the contents of the test tubes by shaking the tubes.

6. Then cover all the test tubes with papers. 7. Place all the test tubes in boiling water bath for

15minutes. 8. Cool the test tubes in cold water and add 5ml

of diluents solvents to each test tube and mix well.

9. Now record the absorbance at 570nm of each solution using a colorimeter.

Then plot the standard curve by taking concentration along X axis and absorbance at 570nm along Y-axis. Estimation of Crude Fibre Crude fibre consists largely of cellulose and lignin (97%) plus some mineral matter. It represents only 60–80% of the cellulose and 4–6% of the lignin. The crude fibre content is commonly used as a measure of the nutritive value of poultry and livestock feeds and also in the analysis of various foods and food products to detect adulteration, quality and quantity. Principle During the acid and subsequent alkali treatment, oxidative hydrolytic degradation of the native cellulose and considerable degradation of lignin occur. The residue obtained after final filtration is weighed, incinerated, cooled and weighed again. The loss in weight gives the crude fibre content. Materials Sulphuric acid solution (0.255 ± 0.005 N): 1.25 g concentrated sulphuric acid diluted to 100 mL (concentration must be checked by titration). Sodium hydroxide solution (0.313 ± 0.005 N): 1.25 g sodium hydroxide in 100 mL distilled water (concentration must be checked by titration with standard acid). Procedure 1. Extract 2 g of ground material with ether or

petroleum ether to remove fat (Initial boiling temperature 35–38°C and final temperature 52°C). If fat content is below 1%, extraction may be omitted.

2. After extraction with ether boil 2 g of dried material with 200 mL of sulphuric acid for 30 min with bumping chips.

3. Filter through muslin and wash with boiling water until washings are no longer acidic.

4. Boil with 200 mL of sodium hydroxide solution for 30 min.

5. Filter through muslin cloth again and wash with 25 mL of boiling 1.25% H2SO4, three 50 mL portions of water and 25 mL alcohol.

6. Remove the residue and transfer to ashing dish (preweighed dish W1).

7. Dry the residue for 2 h at 130 ± 2°C. Cool the dish in desiccators and weigh (W2).

8. Ignite for 30 min at 600 ± 15°C. 9. Cool in a desiccator and reweigh (W3).

CALCULATION % crude fiber in ground sample = Loss in weight on ignition (W2- W1) - (W3- W1) Weight of the sample Observations Antioxidant Power of Rheum emodii Table 1: Free Radical Scavenging Activity of Ascorbic Acid Conc. (μg/ml)

Absorbance of Ascorbic acid

% Reduction

IC50 Value

10 0.292 40.63

26

20 0.269 45.90 30 0.244 50.60 40 0.226 54.45 50 0.202 59.10 60 0.181 63.33 70 0.162 67.21 80 0.141 71.45 90 0.122 75.30 100 0.088 82.16

Graph .1. Straight line Graph Free Radical Scavenging Activity of Ascorbic Acid

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Antioxidant Power Vs Conc. (Ascorbic Acid)





Table 2: Antioxidant Activity of DCM Extract of Rheum emodii Conc. (μg/ml) Absorbance %

Reduction IC50 Value

10 0.280 42.85

50

20 0.274 44.48 30 0.252 46.93 40 0.216 47.55 50 0.201 49.59 60 0.174 53.06 70 0.153 60.26 80 0.141 64.8 90 0.119 69.38 100 0.093 72.44

Graph 2: Antioxidant Activity of DCM Extract of Rheum emodii

Table 3: Antioxidant Activity of Methanol Extract of Rheum emodii Conc. (μg/ml) Absorbance %


10 0.288 43.82

30

20 0.261 46.93 30 0.241 49.38 40 0.230 52.65 50 0.211 57.14 60 0.181 61.22 70 0.162 66.32 80 0.141 71.42 90 0.117 73.26 100 0.072 77.55

Graph 3: Antioxidant Activity of Methanol Extract of Rheum emodii Table 4: Antioxidant Activity of Water Extract of Rheum emodii Conc. (μg/ml) Absorbance %


10 0.310 40.81

42

20 0.291 42.85 30 0.271 44.48 40 0.231 48.97 50 0.215 56.93 60 0.171 64.89 70 0.150 70.81 80 0.131 75.3 90 0.112 78.57 100 0.071 83.26

Graph 4: Antioxidant Activity of Water Extract of Rheum emodii

Table 5: Showing the concentration of various biochemical constituents

S. No Plant extracts Biochemical Constituents

Glucose x 10-3g/l

Protein x 10-3g/l Fibre Ascorbic Acid

x 10-3g/l 1 DCM Extract 159.39 12.5 2.1 0.140 2 Methanol Extract 56.96 65.00 3.2 0.150 3 Water Extract 120.30 53.5 5.2 0.230

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Antioxidant Potential of DCM extract

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0102030405060708090

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Antioxidant Potential of Water Extract





Graph 5: Fiber Content in Various Plant Extracts Graph 6: Data sheet for the Determination of Glucose Graph 7: Glucose Content in Various Plant Extracts

From the Standard Graph the value of (a) as Derived from Beer-Lambert Law comes out to Graph 8: Data sheet for the Determination of Protein Graph 9: Protein content in Various Plant Extracts

From the Standard Graph the value of (a) as derived from Beer-Lambert Law comes out to Graph 10: Data sheet for the Determination of Ascorbic Acid Graph 11: Ascorbic Acid content in Various Plant Extracts

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Fiber Content in Various Plant Extracts

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Standard Graph For Glucose Estimation Concentration Vs Absorbance

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Glucose Content in Various Plants Extracts

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Standard Graph for the estimation of Protein Protein Conc. Vs Absorbance

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Protein Content in various Plant Extracts

A= a. c. t a = 3.3 x 10-3g/L

A= a. c. t a = 2.0 x 10-3g/L





From the Standard Graph the value of (a) as derived from Beer-Lambert Law comes out to Results and Discussion The antioxidant potential of the extracts follows the order ascorbic acid > Methanol Extract > Water Extract > DCM Extract. The glucose content of DCM, Methanol and Water extracts were found to be (159.39, 56.96, 120.30 x 10 -3 g/l). The Ascorbic acid content of DCM, Methanol and Water extracts were found to be (0.140, 0.150, 0.230 x 10-3g/l). The protein content of DCM, Methanol and Water extracts were found to be (12.5, 65.00, 53.5 x 10-

3g/l). The fiber content of the concerned plant extracts was found to be 2.1, 3.2 and 5.2 respectively for DCM, Methanol and Water Extract of the plant. The kingdom Plantae is very interesting, because it furnishes all the types of metabolites which are very useful for the continuity of life, and for good health in addition helps in making our environment clean. The plant in relation showed a good response towards the presence of phytochemicals like, fibers, glucose, protein and ascorbic acid as per results is taken into consideration. It possesses alkaloids, terpenoids, saponins, flavonoids etc, differently in different plant extracts. In addition a study (free radicals, scavenging) activity of the plant were taken into consideration. As per the results, the plant showed a good response towards free radicals scavenging activity. Among the various plant extracts in reference to ascorbic acid, the ethanol extract possess higher antioxidant potential followed by petroleum ether extract. Conclusion As per the result obtained from the concerned plant extracts, it was found that all the three plant extracts bear a good nutritive value. In case where different solvents furnish different nutritive value is due to the interaction of these components with the concerned solvent differently, so different components have different type of

concentrations in the respective solvents, which could be due to polarity index, hydrogen bonding and other mechanisms. From the above results it could be concluded that all the three concerned plants extracts showed a good response towards the fiber content. So the plant in whole should be consumed regularly and as per the bodily demand as they boost up the bodily mechanisms by one way or another way. The secondary metabolites like fiber help in the maintenance of the primary metabolites. The ascorbic acid content, glucose and protein content in the three plant extracts is within the range of consumption, so are useful. References 1. Hina Rehman, Wajeeha Begum, Farzana

Anjum, Humyra Tabasum. 'Rheum emodii (Rhubarb): A Fascinating Herb. 'Journal of Pharmacognosy and Phytochemistry 2014; 3 (2): 89-94.

2. Richa K. Gupta, Gan B. Bajracharya, Ram Narayan Jha. 'Antibacterial activity, cytotoxicity, antioxidant capacity. Phytochemistry 2014; 2 (6): 125-128'.

3. Amandeep Kaur, Sunil Kumar, Ramica Sharma. 'Assessment of Anti-Ulcer Activity of Rheum emodii Rhizomes Extract. Indo Global Journal of Pharmaceutical Sciences, 2012; 2(3): 333-341.

4. Sabreena Rashid, Zahoor A Kaloo, Seema Singh & Iram Bashir''. Medicinal importance of Genus Rheum. International Journal of Advanced Research (2014), 2, (7), 261-267.

5. V Rajkumar, Gunjan Guha, R Ashok Kumar. Apoptosis Induction in MDA-MB-435S, Hep3B and PC-3 Cell Lines by Rheum emodii Rhizome Extracts''. Asian Pacific Journal of Cancer Prevention, 12, 2011.

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Ascorbic Acid Content in Various Plant Extracts

A= a. c. t a = 0.9 x 10-3g/L



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