chemistry project extraction essential oil aniseed

Chemistry Project on Extraction of Essential Oil from Aniseed

Submitted by Editor

Extraction of Essential Oil from Aniseed (Saunf)

Bal Bharati Public School

Ntpc-jhanor

CHEMISTRY INVESTIGATORY

TOPIC:- Extraction of Essential

Oil from Aniseed (Saunf).

NAME:- Siontan Ghosh

CLASS:- XII

SESSION:- 2009-2010

Faculty:- MR. Ashish pagare

CERTIFICATE

This is to certify that Mr. Siontan Ghosh a bonafide student of Class XII has successfully completed the project on “ Extraction of Essential oil from aniseed (Saunf)” in the academic year 2009-2010 .

(Mr. K.T.Johnson)

Incharge Teacher External Examiner

( Mr. Ashish Pagare)

ACKNOWLEDGEMENT

I hereby express my gratitude to my Principal Sri K. T. Johnson and Sri Ashish Pagare for their guidance throughout my studies. I also thank my parents who supported me in all my endeavors. I also thank my classmates who have equally worked hard to make my project a success. I also thank my partner Ayushi Vyas for helping me during the project. And last but not the least I thank the almighty for whatever I have achieved till now.

To extract essential oil present in Saunf (aniseed).

INTRODUCTION

We are all familiar with the pleasant odours coming out from flowers, spices and many trees. The essence or aromas of plants are due to volatile oils present in them. These smelling volatile oils present in plants are called essential oils. Cinnamon, clove, cumin, eucalyptus, garlic, jasmine, peppermint, rose, sandalwood, spearmint, thyme, wintergreen are a few familiar examples of valuable essential oils. The term “essential oils” literally means “oils derived from the essence” of plants.

Essential oils are mainly used for their pleasant odours and flavors in perfumes and as flavoring agents in foods. Some are used in medicines (e.g., camphor, wintergreen, eucalyptus) others as insect repellants (e.g., citronella). Chemically essential oils are composed of complex mixtures of ester, alcohols, phenols, aldehydes, ketones and hydrocarbons. They are essentially non-polar compounds and are thus soluble in non-polar solvents such as petroleum ether, benzene etc. Essential oils may occur in all parts of the plant, but they are often concentrated in the seeds or flowers. They are obtained from the plants by the process of steam distillation and extraction. The technique of steam distillation permits the separation of volatile components from non-volatile materials without raising the temperature of the distillation above 100° C.

Thus steam distillation reduces the risk of decomposition of essential oils.

ANISEED ESSENTIAL OIL

Aniseed Plant

v Aniseed, on steam distillation, yields an essential oil, known as `Oil of Aniseed`, which has now replaced the fruits for medicinal and flavoring purposes. Aniseed oil is a colorless or pale-yellow liquid having the characteristic odor and taste of the fruit. The yield of oil generally varies from 1.9 to 3.1 per cent. Higher values up to 6 per cent have been reported from Syrian aniseed. Crushing of fruits prior to distillation gives better yields of oil. The material should be distilled soon after the crushing to prevent any loss of oil due to evaporation. Aniseed oil is a highly refractive liquid, which solidifies on cooling. The congealing point depends much on the anethole content and is a valuable criterion for evaluating the oil. Exposure of the oil to air causes polymerization, and some oxidation also takes place with the formation of anisaldehyde and anisic acid.

v The chief constituent of aniseed oil is anethole, which is present to the extent of 80 to 90 per cent and is mainly responsible for the characteristic flavor of the oil. The oil also contains methyl chavicol, p-methoxyphenyl acetone, and small amount of terpenes and sulfur containing compounds of disagreeable odour.

Aniseed Essential Oil

v Common Method of Extraction:- Steam Distillation

v Color:- Clear

v Botanical Name:- Pimpinella anisum

v Aromatic Description:- Distinctive scent of licorice. Rich and sweet.

v Constituents:- a-pinene, camphene, B-pinene, linalool, cis-anethole, trans-anethole, safrole, anisaldehyde, acetoanisole.

Uses of Aniseed Oil: -

• Ø In aromatherapy, aniseed essential oil is used to treat colds and flu.
• Ø Aniseed oil can be made into a liquid scent and is used for both hunting and fishing. It is put on fishing lures to attract fish.
• Ø Anethole, the principal component of anise oil, is a precursor that can eventually produce 2,5-dimethoxybenzaldehyde which is can be used in the clandestine synthesis of psychedelic drugs such as 2C-B, 2C-I and DOB.
• Ø Oil of aniseed is also reported to be used as an aromatic carminative to relieve flatulence, and as an ingredient of cough lozenges in combination with liquorice.
• Ø Essential oil is also used externally as an insecticide against small insects such as head lice, mites and vermin. It also has fungicidal properties.

REQUIREMENTS :-

Steam generator (Copper Vessel), round bottom flask (500 ml), conical flask, condenser, glass tubes, iron stand, sand bath, separatory funnel, tripod stands, burners, Ajwain(Carum), Petroleum ether(60-80°C),Saunf(Aniseed) .

PROCEDURE :-

• Set the apparatus as shown in the picture of Experimental Setup. The apparatus consists of a steam generator connected to the round bottom flask through a glass inlet tube. The flask is connected to a water condenser through a glass outlet tube. Condenser is further attached to a receiver through an adaptor.
• Take about 750 ml of water in the steam generator and start heating to produce steam.
• In the round bottom flask take about 75 gm of crushed saunf.
• A vigorous current of steam from steam generator is passed through the round bottom flask.
• A part of the steam condenses in the round bottom flask. As more and more steam is passed, the steam volatile components of saunf pass through the condenser along with steam. These contents on condensation are collected in the receiver.
• The contents in the round bottom flask may be heated by a bunsen burner to prevent excessive condensation of steam.
• The process of steam distillation is continued for about half an hour.
• Transfer the distillate to a separating funnel and extract with 20 ml portions of petroleum ether 3 times.
• Combine the petroleum ether extracts in a 250 ml conical flask and dry it with the help of anhydrous sodium sulphate.
• Remove the solvent from the dried filtrate by careful distillation in a water bath. The essential oil is left behind in the distillation flask.
• Find the weight of the extracted essential oil. Note the colour, odour and weight of the essential oil.

OBSERVATIONS :-

1.) Saunf (Aniseed) :-

Weight of Saunf taken        = 100 gm

Initial Weight of the bottle = 10gm(x)

Weight of bottle + essential oil = 11.25 gm(y)

Weight of essential oil extracted =(y-x) =1.25 gm

Percentage of essential oil = (y/100)*100=1.25 %

Colour of the oil    = Colourless

Odour of the oil = Saunf like smell.

2.) Ajwain (Carum) :-

Weight of Saunf taken        = 75 gm

Initial Weight of the bottle = 10 gm(x)

Weight of bottle + essential oil = 11 gm(y)

Weight of essential oil extracted =(y-x) =1 gm

Percentage of essential oil = (y/75)*100=1.33%

Odour of the oil = Ajwain like smell.

BIBLIOGRAPHY

• Ø Comprehensive Chemistry Practical Class-XII.
• Ø http://en.wikipedia.org/wiki/Anise
• Ø http://www.essentialoils.co.za/essential-oils/aniseed.htm

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Extraction of Essential Oil from Aniseed (SAUNF

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Extraction of Essential Oils Present in Aniseed, Carom

Chemistry Project File Topic-

TO EXTRACT ESSENTIAL OILS PRESENT IN SOUNF(ANISEED),AJWAIN(CAROM) AND

ELAICHI( CARDAMOM )”

Submitted by Submitted to Mr.Arvind Pareek XII A

2 Acknowledgement

I wish to express my deep gratitude and sincere thanks to the principal Mrs. J.Sen Mam, Tagore Public School, Jaipur for her encouragement and for all the facilities that she provided in this project work. I sincerely appreciate this magnanimity by taking me into her fold for which I shall herein indebted to her. I extent my hearty thanks to Mr. Arvind Pareek sir, Chemistry teacher, who guided me to the successful completion of this project . I take this opportunity to express my deep sense of gratitude for his invaluable guidance, attitude and immense motivation, which has sustained my efforts at all stage of this project work

Submitted by Rahul Dugar XII A

3 Certificate

This is to certify that Rahul Dugar has satisfactorily completed the project in chemistry on “TO EXTRACT ESSENTIAL OILS PRESENT IN SOUNF(ANISEED),AJWAIN(CAROM) AND ELAICHI(CARDAMOM)” prescribed by the AISSCE course in the school in the year 2013-14. I have examined the project and hereby accord by my approval of it. As a study carried out and presented in the manner required for its acceptance. This doesn’t necessarily endorse or accept every statement made, opinion expressed or conclusion drawn. It only signifies the acceptance of the project for which it is submitted.

Mrs. J.Sen Mr. Arvind Pareek (Principal) (Head of Chemistry Dept.)

S.NO CONTENTS PAGE NO.

INTRODUCTION 1

EXPERIMENT 2

PROCEDURE AND 4 REQUIREMENTS

OBSERVATION 5 CONCLUSION AND

6 PRECAUTIONS

BIBLIOGRAPHY 7

INTRODUCTION

We are all familiar with the pleasant odours coming out from flowers, spices and many trees. The essence or aromas of plants are due to volatile oils present in them. These smelling volatile oils present in plants are called essential oils. Cinnamon , clove , cumin , eucalyptus, garlic , jasmine, peppermint , rose, sandalwood, spearmint , thyme , wintergreen are a few familiar examples of valuable essential oils. The term “essential oils” literally means “oils derived from the essence” of plants. Essential oils are mainly used for their pleasant odours and flavors in perfumes and as flavoring agents in foods. Some are used in medicines (e.g., camphor, wintergreen, eucalyptus) others as insect repellants (e.g., citronella).

Chemically essential oils are composed of complex mixtures of ester, alcohols, phenols, aldehydes, ketones and hydrocarbons. They are essentially non-polar compounds and are thus soluble in non-polar solvents such as petroleum ether, benzene etc. Essential oils may occur in all parts of the plant , but they are often concentrated in the seeds or flowers. They are obtained from the plants by the process of steam distillation and extraction. The technique of steam distillation permits the separation of volatile components

6 from non-volatile materials without raising the temperature of the distillation above 100° C. Thus steam distillation reduces the risk of decomposition of essential oils.

Experiment To extract essential oil present in  Saunf (aniseed)

 Ajwain(carom)

 Elaichi(cardamom)

Aniseed Essential oil

Aniseed, on steam distillation, yields an essential oil, known as `Oil of

Aniseed`, which has now replaced the fruits for medicinal and flavoring purposes. Aniseed oil is a colorless or pale-yellow liquid having the characteristic odour and taste of the fruit . The yield of oil generally varies from 1.9 to 3.1 per cent. Higher values up to 6 per cent have been reported from Syrian aniseed. Crushing of fruits prior to distillation gives better yields of oil. The material should be distilled soon after the crushing to prevent any loss of oil due to evaporation.

Aniseed oil is a highly refractive liquid, which solidifies on cooling. The congealing point depends much on the anethole content and is a valuable criterion for evaluating the oil. Exposure of the oil to air causes polymerization, and some oxidation also takes place with the formation of anisaldehyde and anisic acid. The chief constituent of aniseed oil is anethole, which is present to the extent of 80 to 90 per cent and is mainly responsible for the characteristic flavor of the oil. The oil also contains methyl chavicol , p-methoxyphenyl acetone, and small amount of terpenes and sulfur containing compounds of disagreeable odour.

9 USES OF ANISEED OIL:-

Ø In aromatherapy, aniseed essential oil is used to treat colds and flu.

Ø Aniseed oil can be made into a liquid scent and is used for both hunting and fishing. It is put on fishing lures to attract fish.

Ø Anethole, the principal component of anise oil, is a precursor that can eventually produce 2,5-dimethoxybenzaldehyde which is can be used in the clandestine synthesis of psychedelic drugs such as 2C-B, 2C-I and

Ø Oil of aniseed is also reported to be used as an aromatic carminative to relieve flatulence , and as an ingredient of cough lozenges in combination with liquorice .

Ø Essential oil is also used externally as an insecticide against small insects such as head lice, mites and vermin. It also has fungicidal properties.

10 Carom essential oil:

Carom seeds are also known as ajwain , Thymol seeds, Onum ,

Ajma , Ajmodika and bishops weed. They are the tiny, cute, delicate and oval shaped herbs with a penetrating fragrance. Carom seed belongs to the family of cumin and parsley . These seeds have been used since years as they consists number of medical properties. Because of their strong aroma, carom seeds are highly used for the Indian culinary.

Carom seeds are sharp and hot with the burning taste, that’s why few seeds are enough to bring the flavor in any Indian recipe. Carom seeds can be whole or powdered one. It is always advisable to buy whole carom seeds as they can be easily turned into powder form just by grinding them in a smooth powder.

USES OF CAROM SEEDS:-

These seeds are used for the tempering or tadka’s in the dishes.

Ajwain has strong, dominant and distinctive taste and flavor, that’s why few seeds are enough to bring exotic fragrance to the vegetarian and non-veg food.

Ajwain can be used for making pickles as well.

The aroma and unique taste of ajwain is used for making various types of roti’s, parathas, thepla’s and so on.

They are specially used in different types of meat, snack recipes and dal recipes to enhance the flavor.

Soups, stocks and stews can be flavored with few seeds of ajwain.

Ajwain are highly incorporated for making various salad dressings and

Indian bakery items.

12 Cardamom Essential oil:

Cardamom provides a warming and stimulating tonic .Cardamom is also an excellent choice for the digestive system. I am sure I don’t have to tell you that Cardamom is greatly admired and extensively used as a culinary spice all over the world. You can also try a gargle with

Cardamon if you suffer with halitosis (bad breath). Why not try 1 drop

Cardamon and one drop of Peppermint in a small amount of water as an effective way to freshen your breath!

USES FOR CARDAMOM

1 Smoothies Add some cardamom to fruit smoothies to give them an extra edge. It doesn’t matter what flavor the basic smoothie is – in fact, the more exotic the better.

2 Fruit loaves If you want to jazz up your banana bread or add a bit of life to your tired fruit loaf recipe, then a touch of ground cardamom will make a world of difference. Just add the spice when you are mixing in the rest of the dry ingredients and you will get a lovely warm, rich flavor from the finished loaf.

3 Bread As with fruit loaves, a sprinkling of cardamom will do wonders for your bread rolls and loaves. Don’t worry about the cardamom turning savory breads into sweet; the spice will just a hint of warmth to the flavor and you can add as little or as much as you like, depending on taste.

Many desserts benefit from the addition of cardamom – rice pudding works especially well, but you could also try adding a little ground cardamom to ice cream, set custards, yogurt, and baked fruit.

5 Casseroles

A few pods added to a casserole dish will give a lamb, chicken or vegetable casserole an extra zing and makes a great entertaining or moving house meal.

14 REQUIREMENTS:-

Steam generator (Copper Vessel), round bottom flask (500 ml), conical flask, condenser, glass tubes, iron stand, sand bath, separatory funnel, tripod stands, burners, Ajwain(Carum), Petroleum ether(60-

80°C),Saunf(Aniseed) .

PROCEDURE:-

Set the apparatus as shown in the picture of Experimental Setup. The apparatus consists of a steam generator connected to the round bottom flask through a glass inlet tube. The flask is connected to a water condenser through a glass outlet tube. Condenser is further attached to a receiver through an adaptor. Take about 750 ml of water in the steam generator and start heating to produce steam. In the round bottom flask take about 75 gm of crushed saunf. A vigorous current of steam from steam generator is passed through the round bottom flask. A part of the steam condenses in the round bottom flask. As more and more steam is passed, the steam volatile components of saunf pass through the condenser along with steam. These contents on condensation are collected in the receiver. The contents in the round bottom flask may be heated by a bunsen burner to prevent excessive condensation of steam.

15 The process of steam distillation is continued for about half an hour.

Transfer the distillate to a separating funnel and extract with 20 ml portions of petroleum ether 3 times. Combine the petroleum ether extracts in a 250 ml conical flask and dry it with the help of anhydrous sodium sulphate. Remove the solvent from the dried filtrate by careful distillation in a water bath. The essential oil is left behind in the distillation flask. Find the weight of the extracted essential oil. Note the colour, odour and weight of the essential oil.

OBSERVATIONS:-

Name of Item Weight Initial Weight Weight Percentage Colour Odour of of Item Weight of bottle of of essential of the the oil taken (x) + essential oil oil essential oil (y/100)*100 oil extracted (y) (y-x)

AUNF 100 gm 10gm 11.25 1.25 gm 1.25 % Colour- Saunf 1.) S gm like -less smell. (ANISEED):-

JWAIN 75 gm 10 gm 11 gm 1 gm 1.33% Colour- Ajwain 2.) A like -less smell (CAROM):-

17 Conclusion

“HENCE WE HAVE EXTRACTED ESSENTIAL OILS FROM 75 GM OF SOUNF(ANISEED),

AJWAIN(CAROM) AND ELAICHI(CARDAMOM)”

Precautions

 If you accidentally spill essential oils – Clean any spillage with an absorbent

material such as kitchen roll.

 If you accidentally get essential oils in your eye – Flush with copious amounts

of MILK for at least 15 minutes and seek medical advice if symptoms persist.

 If you accidentally swallow essential oils – Rinse mouth with MILK and seek

medical attention.

 Handling – Do not eat, drink or smoke when handling. Respect good

personal hygiene.

 Always mix with carrier oil before applying to the skin. Never apply to

inflamed or broken skin.

 Do not use neat on skin.

 Do not take internally. If pregnant seek medical advice before using.

18  Storage – Store in a cool, dry place away from heat and direct sunlight.

Always use original containers. Avoid contact with polished surfaces and

 Keep essential oils away from children and pets

19 BIBLIOGRAPHY

COMPREHENSIVE CHEMISTRY PRACTICAL CLASS-XII. Ø

HTTP://EN.WIKIPEDIA.ORG/WIKI/ANISE Ø

HTTP://WWW.ESSENTIALOILS.CO.ZA/ESSENTIAL-OILS/ANISEED.HTM

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Extraction and Antioxidative Activity of Essential Oil From Star Anise (Illicium verum)

Y. C. Wong * , P. P. Lee and W.A. Wan Nurdiyana

Faculty of Agro Based Industry, Universiti Malaysia Kelantan, Jeli Campus, Locked Bag 100, 17600 Jeli, Kelantan, Malaysia.

DOI : http://dx.doi.org/10.13005/ojc/300329

Star anise ( Illiciumverum ) essential oil was extracted using solvent extraction method. The extraction yields and antioxidant activities of essential oils at different extraction times (1, 3, 5, 7 and 9 days) and temperatures (30, 40, 50, 60, 70 °C) were studied. The results showed that the highest yield of essential oil was 8.56 % by extracting star anise at 60 ⁰C for 7 days.The antioxidant activities of the extracted star anise essential oils were investigated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay on Thin Layer Chromatography (TLC) plates and DPPH radical scavenging method. The results showed that at least two different bands with antioxidant activity with different polarity were appeared on the TLC plates after spraying with DPPH and incubated for 30 minutes. The highest antioxidant activity of star anise essential oil was obtained when the sample was extracted at 60 ⁰C for 1 day (EC 50 value = 0.089±0.05 mg/ml). HPLC analysis showed that the concentration (%) of trans-Anethole present in the essential oils extracted at varied extraction times and temperatures was ranged from 77.29 % to 91.87 %.Gas Chromatography-Mass Spectrometry (GC-MS) analysis was also done on a sample of star anise essential oil and a distinctive peak at retention  time 13.84 minutes with peak area 100% was found to be Estragole compound. Anethole compound was also found to be present at two peaks.

solvent extraction; star anise essential oil; extraction yield; antioxidant activity; trans-Anethole

Introduction

Long term and extensive use of synthetic antioxidant such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) have been proven to cause carcinogenic effects in living organisms [1][2][3]. Due to the potential side effects of synthetic antioxidants, essential oil which derived from organic products can be served as an alternative source for the further improvement of synthetic antioxidant. Star anise ( Illiciumverum ) is a small star-shaped fruit of an evergreen of the Illiciaceae family[4]. This fruit is classified as a spice and it looks like a symmetrical eight-pointed star [5]. Essential oil of star anise has a sweetish, anise flavour and a highly aromatic odour. Due to its high level of phenolic volatile oils, star anise essential oil could be used as a potential alternative antioxidant for synthetic antioxidant.

The objectives of this study were to extract essential oil from star anise using solvent extraction method, to determine the yields and antioxidant activities of essential oils at different extraction times and temperatures, to determine the quantity of trans-Anethole present in star anise essential oil using high performance liquid chromatography (HPLC), as well as tovalidate the presence of Anethole in star anise using Gas Chromatography–Mass Spectrometry (GC-MS).

Materials and Method

Collection of star anise

Star anise dried fruits were purchased from Tong Chun Tang traditional medicine store, Batang Kali, Selangor. The star anise fruits originated from Guangxi province, China. Star anise fruits were stored in plastic bags covered with newspapers to protect them from direct light. Finally, they were kept at room temperature.

Soaking of star anise

50g of dried star anise fruits were weighed using analytical balance (Model: ATX 224, Shimadzu, Japan). The whole star anise fruits were divided into separated carpels and were blended using blender (Model: MX-800S, Panasonic) for 20 seconds. The ground star anise was dissolved in 250 ml of absolute ethanol (ratio of crushed star anise to the ethanol was 1:5, w/v) for a given time (1, 3, 5, 7 and 9 days) at different temperatures (30, 40, 50, 60 and 70 °C).

Extraction of essential oil from star anise

This method was adapted from the work of Thuat and Ngoc (2010) with some modifications [6]. After the extraction time, the solution and solid part were separated by filtration using Whatman filter paper. The brown coloured filtrate was concentrated to volume of 100 ml using rotary evaporator (Model: Hei-vap Precision MLG3B, Germany) at 40 °C. The 100 ml concentrate was mixed with 100 ml of petroleum ether and 100 ml of dH 2 O. The mixture was shook vigorously. After some time of settling, the petroleum ether solution (the upper layer) was dried over anhydrous Na 2 SO 4 . The petroleum ether solvent was removed using rotary evaporator to obtain essential oil. All the essential oils were stored in bijou glass bottles in dark condition at 4 °C before analysis.

Qualitative test using DPPH on TLC

All the star anise essential oils extracted at varied extraction times and temperatures showed antioxidant activities (Table 2). The degree of antioxidant activity of all samples was determined qualitatively from observation of the yellow colour intensity. Based on the yellow intensity of the bands on the chromatograms, the essential oil from star anise extracted at 60 ⁰C and 70 ⁰C for 1 day, 3 days, 5 days, 7 days and 9 days apparently showed stronger antioxidant activity than those extracted at 30 ⁰C, 40 ⁰C and 50 ⁰C.

Besides, it was observed that at least two different bands with antioxidant activity with different polarity were appeared on the TLC plate after spraying with DPPH and incubated for 30 minutes. (Figure 1-5). Samples extracted at 30 ⁰C for five days, 40 ⁰C for three days, 50 ⁰C and 60 ⁰C for nine days had two active bands while the rest of the samples showed three active bands with antioxidant activity (Table 3). The R f values of the active bands in all samples were in the range of 0.41 to 0.65.

Quantitative test using DPPH scavenging assay

The antioxidant activity of star anise essential oil was measured in term of radical scavenging ability, using the stable free DPPH radical. This method was adapted from the work of Brand-Williams, Cuvelier and Berset (1995) with some modifications [8]. Stock solution (1mg/ml) was twofold diluted to different concentrations (0.0625, 0.125, 0.25, 0.5 and 1 mg/ml) with methanol. An aliquot of each dilution, about 1 ml was mixed with 1 ml of 0.004 % methanolic DPPH (0.004 g of DPPH in 100 ml of methanol). The mixture was vortexed and incubated in the dark at room temperature for 30 minutes. The absorbance was measured at 517 nm against a blank (1 ml of methanol with 1 ml of methanolic DPPH without the test oil) using spectrophotometer (Model: Genesys 20 4001/4). The radical scavenging activity or antioxidant activity (%) of each concentration of oil was calculated using the following formula

Antioxidant activity (%)= [ (OD blank – OD assay ) / (OD blank ) ] x 100 %……………………..(Equation 1)

The test was performed in triplicate. The antioxidant activities of the essential oils were expressed as EC 50 , defined as the oil concentration in mg/ml required to scavenge 50 % of the DPPH free radical. BHT was used as positive control.

HPLC analysis

HPLC analysis was performed using Prominence, Shimadzu, Japan. The separation column was Hypersil C18 column (250 mm x 4.6 mm, 5 μm). Essential oil was dissolved in acetonitrile (1 mg/ml). The sample was filtered using syringe with 0.45 µm filters prior to being filled into the vial. 5 ul of sample was injected into HPLC system and the injector temperature was 30 ⁰C. A gradient elution system using mobile phase of 15 %-65 % of acetonitrile with a flow rate of 1 ml/min for 45 minutes was used to identify the compounds present in the star anise essential oil.The detection wavelength was 275 nm.GC-MS analysis of the star anise sample was also done using GCMS 5977A from Agilent Technologies.

Results and Discussion

The extraction yields of essential oils

Maximum yield of star anise essential oil (8.56 %) could be obtained by soaking star anise at 60 ⁰C for 7 days (Table 1). In contrast, extraction of essential oil at temperature of 30 ⁰C for 1 day gave minimum yield of essential oil (4.62 %). It was about 46.03 % less of extraction yield compared to the sample extracted at 60 ⁰C for 7 days.

There was contradictory report from previous researchers. From the previous study done by Ngoc (2006), dried star anise fruits have an essential oil content of 8-10 % [9]. The extraction yield of star anise essential oils in this study was 4.62-8.56 %, which was lower than that of extracted in previous study done by Ngoc. Different extraction yield of essential oil content might be due to several factors such as different geographical locations of the botanical materials, varying in cultivation condition, climate as well as post-harvest factors [10].

Besides, it was observed that the yield of essential oil increased slightly with increasing extraction time and temperature until a certain point. This result was in accordance with Dent and co-workers’ (2012) study, reflected that yield increased with increasing extraction temperature and extraction time [11]. At temperature of 30 ⁰C, the extraction yield increased gradually with days. This result was in accordance with Kumar’s previous study (2010), stated that the longer the extraction time, the longer the contact time of the plant material with the solvent and hence more yields of complete oil [12]. For temperature of 40 ⁰C and 70 ⁰C, the extraction yields decreased when star anise soaked for more than 5 days. Same behavior was observed at temperature of 50 ⁰C and 60 ⁰C, the yields decreased with an increase in extraction time at 9 days. This may be due to the loss of solvent by evaporation when extraction time and temperature increased simultaneously during extraction.

For star anise soaked for 1, 3 and 7 days, the yield of essential oil increased from 30 ⁰C to 60 ⁰C. The extraction yield decreased with a further increase in temperature at 70 ⁰C due to loss of solvent and volatile oil at high temperature. The increase of extraction yield with increasing extraction time and temperature may be due to increased solubility and diffusion coefficient [11]. In this present study, the extraction temperature of 60 ⁰C and the extraction time of 7 days were the best condition to increase the extraction efficiency of star anise essential oil. It is therefore suggested that an extraction temperature of no higher than 60 ⁰C is employed.

The radical scavenging activity or antioxidant activity (%) of each concentration of oil and BHT were calculated using the formula (Equation 1) and the results were shown in Table 4. Generally, all the samples extracted at 30 ⁰C for 1, 3, 5, 7 and 9 days showed low antioxidant activity, which was below 60 %. Figure 6 showed that the antioxidant activity (%) of essential oils extracted at 30 ⁰C increased steadily with increasing concentration from 6.25 x 10 -3 mg/ml to 1.00 mg/ml. The antioxidant activity of each sample was then expressed as EC 50 , which is the concentration of sample required to decrease the initial DPPH free radical by 50 %.

It was observed that star anise essential oil extracted at 30 ⁰C for 1 day had the greatest EC 50 value of 0.973 mg/ml (Table 5). This means that the essential oil extracted at 30 ⁰C for 1 day had lowest antioxidant activity as it required 0.973 mg/ml to scavenge 50 % of the free DPPH radicals. The EC 50 values for samples extracted at 30 ⁰C for 3 days (0.967 mg/ml), 5 days (0.928 mg/ml), 7 days (0.893 mg/ml) and 9 days (0.622 mg/ml) were declined gradually. This indicated that the degree of antioxidant activity increased with increasing extraction time at 30 ⁰C. This might be due to longer contact time of star anise with solvent and hence increased the rate of extraction of potential active compounds with antioxidant activity [12]. The ascending order for the antioxidant activity was 1 (30 ⁰C) ˂ 3 (30 ⁰C) ˂ 5 (30 ⁰C) ˂ 7 (30 ⁰C) ˂ 9 (30 ⁰C).

The antioxidant activity (%)  of all the star anise essential oil extracted at 40 ⁰C  was above 60 % (Table 4 and Figure 7). The EC 50 value for essential oil extracted at 40 ⁰C for 1 day was 0.585 mg/ml (Table 6). The EC 50 value dropped to 0.391 mg/ml when the essential oil extracted at 40 ⁰C for 3 days. After that, there was an increase in EC 50 values until day 9. The increase of EC 50 values corresponded to a weaker antioxidant.The order of antioxidant capacity for samples extracted at 40 ⁰C was 1 (40 ⁰C) < 9 (40 ⁰C) < 7 (40 ⁰C) < 5 (40 ⁰C) < 3 (40 ⁰C).

In general, the antioxidant activity (%) of star anise essential oils extracted at temperature of 50 ⁰C was higher than 70 % (Table 4 and Figure 8). According to Table 7, essential oil extracted at 50 ⁰C for 1 day showed EC 50 value of 0.098 ± 0.06 mg/ml. The concentration of essential oil needed to scavenge 50 % of DPPH radical increased from day 1 to day 7. However, there was a marked decrease of concentration of essential oil to scavenge 50 % of the radicals. The antioxidant power of sample extracted at 50 ⁰C for 9 days (0.099 ± 0.06 mg/ml) was as good as the sample extracted for 1 day. In other words, essential oil extracted at 50 ⁰ C for 1 day was the strongest antioxidant, followed by those essentail oil extracted at 9 days, 3 days, 5 days and 7 days.

All the essential oils extracted at 60 ⁰C showed antioxidant activity greater than 70 % (Table 4 and Figure 9). The results in Table 8 showed that essential oils extracted at 60 ⁰C for 1 day was the strongest antioxidant. This was because only 0.089 ± 0.05 mg/ml of essential oil was needed for the scavenging activity of free radicals. The antioxidant power was decreasing in the order 1(60 ⁰C) > 5(60 ⁰C) > 7(60 ⁰C) > 3 (60 ⁰C) > 9 (60 ⁰C).

Compared with essential oil extracted at 60 ⁰C for 1 day (EC 50 value= 0.089 ± 0.05), essential oil extracted at 60 ⁰C for 9 days (EC 50 value= 0.583 ± 0.34) showed a significant difference in term of concentration in mg/ml necessary to scavenge 50 % of the DPPH radicals. The antioxidant activities of star anise essential oils extracted at 60 ⁰C were undulated with increasing extraction time. This might be due to the possible synergistics effects of different compounds on the total oil antioxidant activity [13].

All the star anise essential oils extracted at 70 ⁰C for varied extraction time showed antioxidant activity (%) above 70 % (Table 4 and Figure 10). Since lower EC 50 value indicated higher antioxidant activity, essential oil extracted at 70 ⁰C for 3 days exhibited stronger antioxidant activity than essential oil extracted for 1 day. The EC 50 value for essential oil extracted for 3 days and 1 day were 0.100 ± 0.06 mg/ml and 0.135 ± 0.08 mg/ml respectively as shown in Table 9. Essential oils extracted more than 3 days, which were 5 days, 7 days and 9 days showed decreasing of antioxidant power as the EC 50 value increased markedly from 0.100 ± 0.06 mg/ml to 0.310 ± 0.18 mg/ml. The antioxidant activity was in order 3 (70 ⁰C) > 5 (70 ⁰C) > 7 (70 ⁰C) > 1 (70 ⁰C) > 9 (70 ⁰C).

There was an increase in antioxidant activities of star anise essential oils from 30 ⁰C to 50 ⁰C. This might due to ambient or physiological temperatures could reduce thermal degradation of volatile essential oil [14]. The antioxidant activities of star anise essential oils decreased slightly at 60 ⁰C. This was in agreement with Dent and research group (2012), stated that degradation of phenolic compounds may occured at temperature of 60 ⁰C [11]. At temperature 70 ⁰C, the antioxidant activities of star anise essential oils increased moderately. Typically, antioxidant activity decreased with increasing temperature, but it does not have universal validity [15].

In this quatitative antioxidant test using DPPH method, a well known synthetic antioxidant, namely butylated hydroxytoluene (BHT) was used as positive control. The concentration of BHT for a 50 % scavenging activity was about 0.018 mg/ml [16]. In this study, 0.016 ± 0.01 mg/ml of BHT was required in scavenging action towards DPPH free radicals (Table 10).By comparing the antioxidant activity of star anise essential oil with BHT, it was revealed that antioxidant activity of essential oil from star anise was comparable with the synthetic antioxidant BHT, particularly essential oils that were extracted at temperature 50 ⁰C and 60 ⁰C.

HPLC and GC-MS analysis

The HPLC chromatogram of standard trans-Anethole with retention time of 35.487 minutes was shown in Figure 11. It was observed that the highest peaks of all samples have a retention time near to the retention time of standard trans-Anethole, which was around ±35 minutes. Similarities of the highest peak and retention time between samples and standard trans-Anethole in the HPLC chromatogram confirmed that trans-Anethole was present in star anise essential oil. In order to identify the concentration (%) of trans-Anethole present in all the essential oils, a standard curve of trans-Anethole was plotted (Figure 12). The retention times, peak areas, area percentage and concentration (%) of trans-Anethole for all samples were shown in Table 11.

All the star anise essential oils had high area percentage of trans-Anethole, which was ranged from 77.29 % to 91.87 %. High percentage area indicated trans-Anethole was the most abundant compounds in all the star anise essential oils. Similar results were reported previously by Chempakam andBalaji (2008), stated that the main component of star anise essential oil is trans-Aanethole, which accounts for 80-90 % [4].  From GC-MS analysis, Estragole was found to have the most significant peak with peak area 100% at retention time 13.840 minutes as shown in Figure 13 and 14 and Table 12.Estragole was found at the tenth peak.Anethole was also found at two peaks which were peak 11 and 12 as shown in Figure 15 and Figure 16. Thus, the data validates the presence of Anethole in the star anise sample.

In addition, it was observed that the concentration (%) of trans-Anethole in the star anise essential oils varied considerably as function of temperature and extraction time. Generally, star anise essential oils extracted at 40 ⁰C, 50 ⁰C, 60 ⁰C and 70 ⁰C contained relatively high concentration (%) of trans-Anethole compared to essential oils extracted at 30 ⁰C. As temperature increases, higher mass fraction of trans-Anethole was extracted. It was in agreement with Chen and co-workers’ finding (2007), which concluded that higher temperature will reduce the solvent viscosity and facilitate the diffusion of molecules, resulting to an increase of extraction efficiency [17].

Besides, it was found that the concentration (%) of trans-Anethole corresponded to the antioxidant activities of star anise essential oils. This statement was in agreement with Padmashree and research group (2007), reported that the antioxidant activity is due to high percentage of trans-Anethole, which is more than 80 % [18]. In the present study, star anise essential oil extracted at 60 ⁰C for 1 day has the highest concentration of trans-Anethole (91.61 %) and antioxidant activity (EC 50 =0.089 mg/ml) while star anise essential oil extracted at 30 ⁰C for 1 day has the lowest concentration of trans-Anethole (45.03 %) and antioxidant activity (EC 50 =0.973 mg/ml). Therefore, it might be possible to infer that trans-Anethole found in the samples contributed to the antioxidant activities of the essential oils.

Essential oil from star anise was successfully extracted using solvent extraction method. Different extraction times (1 day, 3 days, 5 days, 7 days and 9 days) and temperatures (30 ⁰C, 40 ⁰C, 50 ⁰C, 60 ⁰C and 70 ⁰C)did affect the yield of essential oil. The best condition to obtain the highest yield (8.56 %) of essential oil was at temperature of 60 ⁰C with an extraction time of 7 days. A rapid screening using DPPH assay on TLC showed thatat least two different yellow spots with antioxidant activity with different polarity were appeared on all TLC plates at solvent system 95:5, v/v toluene/ ethyl acetate. In addition, the highest antioxidant activity of star anise essential oil was observed when the sample was extracted at 60 ⁰C for 1 day (EC 50 value = 0.089±0.05 mg/ml).From the HPLC analysis results, samples with higher trans-Anethole concentration have higher antioxidant activities. Star anise essential oil extracted at 60 ⁰C for 1 day showed the highest concentration (%) of trans-Anethole (91.61 %) while essential oil extracted at 30 ⁰C for 1 day showed the lowest concentration (%) of trans-Anethole (45.03 %). The antioxidant activity of the high level of trans-Anethole-containing star anise essential oil was comparable with the synthetic antioxidant BHT.

Acknowledgement

The authors express their sincere appreciation to Faculty of Agro Based Industry, Universiti Malaysia Kelantan, Jeli Campus for the financial support which made this study possible.

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