asian journal of biochemical and pharmaceutical research

Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol. 1) 2011 ISSN: 2231-2560

Research Article

410

Asian Journal of Biochemical and Pharmaceutical Research

Microwave Solvent Extraction (MSE) as an Effective Technique Against Traditional Solvent Extraction (TSE) for Screening Different Plant Extracts for Antioxidant

Activity

Abhishek Mathur*1, 2, 3, Deepika Mathur4, GBKS Prasad3 and V.K. Dua1

1National Institute of Malaria Research, Hardwar (U.K), India.

2Dev Bhoomi Group of Institutions (DBGI), Dehradun (U.K), India.

3Jiwaji University, Gwalior (M.P), India

4Jawaharlal Nehru Cancer Hospital & Research Centre, Bhopal (M.P), India.

Received: 12 April 2011; Revised: 6 May 2011; Accepted: 16 May 2011

Abstract: In the present investigation 18 plants belonging to 12 families were randomly selected and different solvent extracts were prepared both by MSE and TSE of different plants and were evaluated for their in vitroantioxidant activity. The MSE of the each of the plant material was performed at 800C in Teflon vessels of the Ethos E. Microwave Extraction System (Milestone, Inc. Monroe, CT) while TSE was performed by conventional method. The methanol extracts prepared by MSE method showed maximum yield in comparison to other solvent extracts prepared by MSE and TSE. The plant extracts were then after evaluated for their in vitro antioxidant activity by DPPH assay, Superoxide Anion radical scavenging Activity and Total Phenolic Content (TPC). The results indicate that extracts prepared by MSE technique showed potent antioxidant activity in comparison to the extracts prepared by TSE method.

Keywords: Plant extracts, antioxidant activity, MSE, TSE, DPPH assay, Superoxide Anion Radical Scavenging Activity, TPC.

INTRODUCTION:

India is one of the nations blessed with a rich heritage of traditional medical systems and rich biodiversity to complement the herbal needs of the treatment administered by these traditional medical systems. The recognized Indian Systems of Medicine are Ayurveda, Siddha and Unani, which use herbs and minerals in the formulations. The Health advantages of diets rich in antioxidant plant compounds include lowering the risk of cardiovascular disease, certain cancers and the natural degeneration of the body associated with the aging process. Free radicals are unstable molecules formed when the body uses oxygen for energy. The instability of these molecules can damage tissues, alter DNA and change cell structure. Ultimately, free radicals start a chain reaction resulting in the reproduction of even more free radicals. Antioxidants interact with and stabilize free radicals and may prevent some of the damage, free radicals cause to the body. The role antioxidants have in free radical stabilization involves the antioxidants donating one of their own electrons to the free radical. This electron donation is done without the antioxidant becoming unstable or damaging to the body. This

Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol. 1) 2011

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remarkable action stabilizes the free radicals as quickly as they are produced in the human body. Recently, natural plants have received much attention as sources of biological active substances including antioxidants. Numerous studies have been carried out on some plants, vegetables and fruits because they are rich sources of antioxidants, such as vitamin A, vitamin C, Vitamin E, carotenoids, polyphenolic compounds and flavanoids [1] which prevents free radical damage, reducing risk of chronic diseases. Thus, the consumption of dietary antioxidants from these sources is beneficial in preventing cardiovascular disease [2]. Antioxidant activity in peel and pulp of Citrus fruits, different varieties of Apple (Pyrus malus L.) of Kashmir (J&K) and different plants of Uttarakhand were investigated [3-5]. The search for newer natural antioxidants, especially of plant origin has ever since increased. In this study, the Traditional solvent extraction (TSE) and Microwave solvent extraction (MSE) methods were compared for extraction of high yield crude extract in different plant solvent extracts. The results can determine the natural antioxidants available in the plant parts having solubility in the specific solvent. Also, the extraction methods will emphasize on using the specific solvent (methanol, water, petroleum ether and hexane) for extracting antioxidants and polyphenolics. This study may provide insight for future extraction solvents and natural potent antioxidants which can be used as dietary supplement.

MATERIALS AND METHODS:

The chemicals and reagents used in the study were from Ranchem. & CDH. The glass wares used were from Borosil. The MSE method comprises the use of Ethos E. Microwave Extraction System (Milestone, Inc. Monroe, CT).

Collection of material

The plant material was collected from the specific plant. The plants were authenticated by Botanical Survey of India (BSI), Dehradun (U.K), India. The plant material was dried under shade and was ground to form the fine powder.

Traditional Solvent Extraction (TSE)

Methanol, water, petroleum ether and hexane were the solvents used for the conventional solvent extraction. Conventional TSE method was followed [6]. About twenty grams of each of the powdered plant material was soaked separately in different solvents according to decreasing polarity for about 72 h. After every 24 h, the mixture was stirred with the sterile glass rod. At the end of 72 h, the beakers having the mixtures were capped and placed in a 600C water bath for 20 minutes. These beakers were vortexed twice during the incubation. Then, the solvent layer from each test tube was separated by centrifugation at 2000 rpm for 15 minutes. The solvent supernatants were transferred to clean, previously weighed and labeled beakers/ petridishes. The residue was mixed with 20 ml of the same solvent again and vortexed. The solvent supernatants were combined with the previous one. The beakers/ petridishes with supernatant were then placed in a vacuum evaporator to remove solvent. The dried extracts in the beakers/ petridishes were weighed to measure the extraction yield of the samples. All samples were placed in a freezer set at – 200 C prior to testing.


412

Microwave assisted Solvent extraction (MSE)

Twenty grams of the plant material was weighed separately using a clean aluminum container and transferred into each of the Teflon vessels of the Ethos E Microwave Extraction System (Milestone, Inc. Monroe, CT). The Teflon vessels were covered with polymer material that can resist high inside pressure generated when extraction temperatures are higher than the used solvent’s boiling point. Specific solvent (40 ml), as used above in TSE were used and a magnetic stirring rod were added in each vessel for each of the plant material. The vessels were sealed and properly placed inside the Microwave Extraction System. Then, the Microwave Extraction System was programmed to increase to the extraction temperature with a maximum energy level of 800 Watts and held at that temperature for 20 min with an energy level of 500 Watts. Extraction temperature 80 o C was applied to perform the microwave-assisted solvent extraction, respectively. After a twenty-minute cool down period, the vessels were unsealed and transferred to each corresponding centrifuge tube. These tubes were centrifuged at 2000 rpm for 15 minutes to separate the supernatant and residue. The solvent supernatant was transferred to a clean test tube that had been previously weighed. The residues were mixed with 20 ml of same solvent again and vortexed. The solvent supernatant was separated by the centrifugation and combined with the previous one. The supernatant was then placed in a vacuum centrifuge evaporator to remove the solvent. The dried extract in the test tube was weighed to measure the extraction yield of the samples. The samples were stored in a -20oC freezer prior to testing.

Determination of Total Phenolic Content (TPC)

The Total Phenolic Content of each fraction obtained of each of the plant material was determined [7] and the phenolic content was expressed as µg/g Gallic acid equivalents. In brief a 100 µl aliquot of the sample was added to 2 ml of 0.2% (w/v) Na2CO3 solution. After two minutes of incubation, 100 µl of 500ml/l Follin-Ciocalteu reagent added and the mixture were then allowed to stand for 30 minutes at 250C. The absorbance was measured at 750 nm using a UV-VIS Systronics spectrophotometer. The blank consist of all reagents and solvents but no sample. The Total Phenolic Content (TPC) was determined using the standard Gallic acid calibration curve and was expressed as µg/g Gallic acid equivalents.

Determination of Antioxidant Activity by DPPH Radical Scavenging Method

The extract solution for the DPPH test [8] was prepared by re-dissolving 0.2 g of each of the dried extract in 10 ml of the specific solvent in which the extract was prepared. The concentration of DPPH solution was 0.025 g in 1000 ml of methanol. Two ml of the DPPH solution was mixed with 10, 20 and 40 µl of the plant extract solution and transferred to a cuvette. The reaction solution was monitored at 515 nm, after an incubation period of 30 minutes at room temperature, using a UV-Visible Systronics spectrophotometer. The inhibition percentage of the absorbance of DPPH solution was calculated using the following equation: Inhibition%= (Abst=0 min -Abst=30 min)/ Abst=0 min ×100 Where Abst=0 min was the absorbance of DPPH at zero time and Abst=30 min was the absorbance of DPPH after 30 minutes of incubation. Ascorbic acid (0.5 mM) was dissolved in methanol and used as a standard to convert the inhibition capability of plant extract solution to the Ascorbic acid equivalent. IC50 is the concentration of the sample required to scavenge 50% of DPPH free radicals.


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Superoxide Anion Radical Scavenging Activity

Superoxide Anion Radical scavenging Activity was measured [9] with some modifications. The various fractions of plant(s) extracts were mixed with 3 ml of reaction buffer solution (pH, 7.4) separately containing 1.3 µM riboflavin, 0.02 M methionine and 5.1 µM NBT. The reaction solution was illuminated by exposure to 30W fluorescent lamps for 20 minutes and the absorbance was measured at 560 nm using Systronics UV-VIS double beam spectrophotometer. Ascorbic acid was used as positive control and the reaction mixture without any sample was used as negative control.

The Superoxide anion radical scavenging activity (%) was calculated as:

RESULTS AND DISCUSSION:

From the present investigation it is confirmed that MSE methods are much more efficient than TSE methods for extracting the better yield of antioxidants. The results are given in Figure 1. The present study illustrate that methanol solvent is best suited for extraction of antioxidants. It was found that methanol solvent extracts produced by MSE showed maximum antioxidant potential in comparison to other solvent extracts (results of antioxidant activity of other solvent extracts are not shown). The Total Phenolic Content (TPC) was found to be maximum in Syzigum cuminii (seeds) followed by Ainsliaea apetra (leaves), Bacopa monnieri (whole plant) and Mentha piperita (root). The results of TPC are recorded in Table 1.

The results of Superoxide Anion Radical Scavenging Activity were recorded as % inhibition of superoxide and are shown in Table 3. The results are totally correlated to each other and confirm the use of these plants as natural antioxidants. Presence of polyphenolics in the extract confirm their utility as potent antioxidant agent as revealed by the experimental results.

CONCLUSION:

The present study thus leads to the conclusion that MSE method can be utilized as an efficient technique against TSE method for isolation and extraction of natural antioxidant agents/secondary metabolites. However further studies are needed to isolate and characterize the active molecule present in each of the plant extract responsible for antioxidant activity. These studies may further leads to the formulation of a new drug having the active constituent present in each of the plant extract.


414

0102030405060708090

Percent yield

A. milleof. L.cephalotes S. chirayta

Plants

Percent yield of methanol extracts prepared by different techniques

TSE

MSE

Fig. 1. Percent yield of methanol extracts of plants prepared by different extraction methods


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Table 1. TPC of methanol extract prepared by MSE of different plants

Sr. No. Plants Total Phenolic Content (TPC) µg/g Gallic acid

Microwave Solvent Extraction (MSE)

1. A. milleofolium 220.0

2. A. aspera 250.0

3. A. apetra 224.0

4. Aloe vera 226.0

5. A.racemosus 215.5

6. B.monnieri 241.3

7. C. nurvula 220.6

8. G. superba 210.0

9. L.cephalotes 235.8

10. M. borealis 224.0

11. M. piperita 241.3

12. M.koenigii 226.0

13. P.kurroa 216.0

14. R.pulchellus 65.23

15. R.communis 137.8

16. R. cinerea 220.6

17. S. chirayta 237.2

18. S. cuminii 250.6


416

Table 2. IC50 of methanol extract prepared by MSE of different plants by DPPH assay

Sr. No. Plants DPPH assay-IC 50 values (μg/ml)



2. A. aspera 30.10

3. A. apetra 25.10

4. Aloe vera 25.00


6. B.monnieri 46.00

7. C. nurvula 33.10

8. G. superba 25.00





13. P.kurroa 42.00







417

Table 3. Percent inhibition of superoxide of methanol extract prepared by MSE of different plants by

Superoxide Anion Radical Scavenging Activity

Sr. No. Plants Superoxide Anion Radical Scavenging Activity (%)



2. A. aspera 72.00

3. A. apetra 72.00

4. Aloe vera 75.50


6. B.monnieri 65.68

7. C. nurvula 72.30

8. G. superba 75.50





13. P.kurroa 80.10







418

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*Correspondence Author: Abhishek Mathur, Dev Bhoomi Group of Institutions (DBGI), Dehradun

(U.K), India.

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