Different methods of extraction for Red Dye from Capsicum annuum

 

Sachin Aglawe*, Amol Gayke, Prasad Murade, Prashant Swami, Pramod Game, Pratik Kadam, Shubham Kanawade, Yogesh Garud

 

Abstract:

Natural dyes produce an extraordinary variety of products and complex colors that complements each other. Straight off a day synthetic leads to high environmental pollution. The present investigation was carried out to extract natural dye from green chili (Capsicum annum). The main coloring component in chili is oleoresin. The dye was extracted using solvent extraction method. A mordant is a substance used to set dyes on fabrics by forming a coordination complex with the dye which then attaches to the fabric. Capsicum red pigment extracted from the dry pepper is a kind of high-quality natural dye which has anticancer and cosmetic properties. The variety used to manufacture paprika extract for food colouration is Capsicum annuum. Colour extracts have a very low content of capsaicin in contrast to the extracts used as flavouring agents. Plant pigments represent a source of non-toxic compounds that are used as food or cosmetic coloring agents. Red-fruited varieties of Capsicum annuum can be extracted to isolate the red-colored xanthophylls, capsanthin, and capsorubin. Common commercial processes for this extraction use hexane as the extracting solvent and mild or no heat varieties of Capsicum. The simple spectrophotometric method of determination of capsaicinoids in fresh fruit of hot pepper and the HPLC method were compared. Capsaicinoids were extracted from hot pepper fruit with the petroleum ether: acetone mixture, and then separated using thin layer chromatography on silica gel and evaluated quantitatively using the HPLC method and spectrophotometrically.

 

 

 

 

INTRODUCTION:

Natural dyes, which were pushed during the last sixty years into the background by synthetic dyes, are recently again becoming object of consumer interests. This is due to the awareness of possible risks during production of synthetic dyes and also when used on textiles they can cause allergy, skin disease, cancer etc. Some of the synthetic dyes which are even carcinogenic and mutagenic have been banned. So, there is need to produce natural dyes. Natural dyes have been shown to possess health- promoting properties. Natural dyes are obtained from natural sources such as plants, insects and minerals. Among all the plant based dye sources i.e. bark, leaves, flowers, seeds etc. So, In this review we are focusing on comparative study on various extraction methods used for the extraction of coloring compound (dye) obtained From dried fruits of Capsicum annuum by various eco-friendly and less harmful methods.

 

Biological Source:

Capsicum consists of dried fruits of Capsicum annuum

 

Family: Solanaceae

 

Chemistry:

Capsaicin: a phenolic compound (0.02%) is the major component of the oils present and is mainly responsible for the pungency of the drug. The other molecule responsible for pungency as well in lesser fractions is 6, 7-dihydrocapsaicin. Capsicum also contains around 1.5% of oleoresin. Capsianosides are also present.

 

Structure 1: Capsaicin

 

Structure 2: Capsianosides

 

Chilies also contain thiamine, ascorbic acid, capsanthin and capsorubin which are carotenoids and fixed oils. The Hungarian variety is a good source of Vitamin C.

 

Method-1: Extraction of capsicum red pigment

Materials:

Source:

Green chili was collected from Market Yard, Nashik.

 

Reagents:

Ethanol, Sodium hydroxide, Hydrochloride, Leveling agent FFA, Penetrating agent JFC.

 

Instruments:

Soxhlet extractor, High-speed universal pulverizer. Electric blast drying oven, electric heating-jacket, thermostat water bath, Electronic balance, visible spectrophotometer, Electric thermal-vacuum drying oven [1]

 

Procedure:

Dry capsicum powder was transferred to a Soxhlet extractor which was added into 95% ethanol. To ensure the optimal extraction process as given in table no: 1. finally, head product was obtained after vacuum distillation and recovering ethanol.

 

Table 1 Different optimum condition of extraction:

Temperature (in 0C)	Solid Liquid Ratio	Reflux Time (min)
30	1:5	30
40	1:10	60
50	1:15	90
60	1:20	100
70	1:25	110
80	1:30	120
90	1:35	150

 

Purity of capsicum red pigment:

Silica gel column chromatography was selected to purify capsicum red pigment: elution condition was censured by thin layer chromatography. First, concentration extracted was injected into the column with 100cm in high, 10cm in diameter and was eluted by eluent selected i.e. ethanol. Afterward, several parts of solution were gained and red part was collected and vacuum- concentrated to relatively pure capsicum red pigment. At last, the capsicum red pigment was analysed by methods of infrared spectrum (IR). [1]

 

Method–2: Extraction of Natural Dye from Chili (Capsicum Annum)

Materials:

Source: Red chili was collected from Market Yard, Nashik.

 

Reagents:

Laboratory grade chemicals- 95% ethanol, coppersulphate and ferrous sulphate.

 

Instruments:

Weighing balance, Water bath, Soxhlet apparatus, Hot air oven, Colorimeter, UV Transilluminator.

 

Method:

Preparation of Raw Material:

The samples were collected and washed thoroughly with water to remove any impurities. After drying at room temperature, the samples were ground into powder with the help of grinder. [2]

 

Extraction of Crude Dyestuff:

100 g of sample was weighed and taken in a round bottom flask and 500ml of solvent (ethanol water) was added to it. The flask was heated in a water bath at 60°C for 60mins.The solution was then filtered to obtain crude dyestuff. [2]

 

Purification of Crude Dyestuff:

The crude dyestuff is distilled to get 1/3^rd of the solution using the Soxhlet apparatus at 70°C for 3hrs, ethanol is recovered and the concentrated dye is obtained. The solution is kept overnight at room temperature for precipitation. The precipitation in ethanol water is obtained by decanting the solution. The obtained particles are dried inanoven overnight at 60°C. Water was added in the soxhlet apparatus. By addition of water, the boiling points of the compounds are lowered, allowing them toevaporate at lower temperatures. [2]

 

Method-3:

Materials:

Source: Red chili was collected from Market Yard, Nashik.

Reagents: Deionized water.

 

Instruments:

Weighing balance, Volumetric Flask, Hot air oven, heating mantle.

Method:

The fruits of red chili (Capsicum annum L.) were collected from Nashik region. The collected plant materials were used for extraction ofdye. The cleaned samples (50g) were crushed, dissolved in deionized water (500 ml) and then boiled for 2 hours in a hot water bath for quick extraction. At the end of 2 hours, the total color was extracted. The solution was then double filtered. The obtained extract is dried in an oven overnight at 60 ⁰C. [3]

 

Method-4:

Materials: Hot pepper Capsicum annum L. Capsaicin, TLC plates (Silica gel G).

Reagents: Na₂SO_4, Mixture of acetone and petroleum ether (1:1), acetone, petroleum ether: acetate: methanol (75:20:5), 2, 6-dichloro-chinonechloroimide.

 

Instruments:

Homogenizer, separatory funnel, HPLC.

 

Methods:

Extraction:

Samples of hot pepper pericarps (10 g of dried material) were homogenised with acetone and then with a mixture of acetone and petroleum ether (1:1) until the tissue was decolorized. The extract was filtered under reduced pressure and was put into a separatory funnel where acetone was washed out with small portions (0.01 ml) of water. The obtained ether layer was dried with anhydrous Na₂SO₄ and concentrated with vacuum evaporator at 30⁰C. [4]

The extraction procedure is presented in Fig. 1.

 

Figure -1 Flow diagram for extraction of capsaicinoids

 

Isolation and identification of capsaicinoids:

The ether extract (1 ml) was put on the TLC plate and developed in petroleum ether:acetate: methanol (75:20:5). Three plates with purified extracts and one with capsaicin standard (0.4 ml, 0.2 mg/ml) were developed in parallel. In order to locate the capsaicinoids on the plate, one of them was sprayed with the solution of 2, 6-dichloro- chinonechloroimide. The Rf values were determined for capsaicinoids. The bands corresponding to cap-saicinoids were scraped off the plates not being sprayed and the compounds were washed out of silica gel with methanol. They were used for colorimetric and HPLC determination. [4]

 

Method -5:

Materials:

Plant material:

Capsicum annuum: Entire fruits were dried for 48 to 72 h at 47⁰C in an oven and then the fruit were ground to a powder using grinder. Dried ground pungent hot-tasting red powder (cayenne) was purchased at local retail sources.

 

Reagents:

20% ethanol, 4800 psi CO_2, ethanol, water, hexane, methanolic KOH, N₂ gas, CHCl_3.

 

Instruments:

ISCO SFX 3560, Neslab RTE-101, centrifugation apparatus,Dionex ASE 350, vortexer.

 

Method:

Extractions:

SFE was performed using an ISCO SFX 3560 Supercritical Fluid Extractor with a Neslab RTE-101 recirculating water chiller. The final optimal conditions were up to 3 g dried, ground fruit was placed in the thimble and extracted with 20% ethanol (95%) as a modifier for 5 min static, 20 min dynamic at 60 ⁰C, 4800 psi CO₂ (0.86 g/ml); we removed the modifier and re-extracted, 5 min static, 20 min dynamic at 60⁰C, 4800 psi CO₂; and finally, the restrictor was washed with 5 ml ethanol. This resulted in 7 ml of an ethanol extract in the collection tube for each thimble extracted. The ethanol is considered a modifier, because it was present during the first extraction cycle withsupercritical CO₂. The restrictor is the metal tube that dispenses the extract from the pressurized thimble. This tube was washed with ethanol to rinse out all of the extract. The restrictor was washed between samples, and blank extraction thimbles were placed between each sample thimble to prevent any carryover between samples. The sample was then adjusted to 76% ethanol by the addition of 1.4 ml water. The samples were placed on ice for 30 min and then centrifuged 0 ⁰C, 4000 rpm, for 20 min. The supernatant and the pellet were collected separately. For characterization, the pellet was resuspended in hexane, typically 5 ml. Two different hexane extractions were performed. The first was an overnight batch extraction with hexane, 1 g dried commercial cayenne powder with 70 ml, hexane for 16 hrs at room temperature. The hexane was decanted and the oleoresin dried under a stream of nitrogen. The weight of the carotenoids extracted by the hexane was determined. The second hexane extraction was performed using a Dionex ASE 350. In this case, 3gm of dried ground red Capsicum fruit placed in a 5-ml steel thimble and extracted with hexane with the following settings: 8 min static, 40 ⁰C, 1500 psi; hexane-extracted samples were dried under N₂ gas to reduce the final volume to 5 ml. The samples were saponified, It was used with the following modifications: 300 ml of carotenoids in hexane were mixed with 300 ml methanolic KOH for 1 h at 50⁰C with frequent vortexing; and the free carotenoids were separated from the acyl groups with the addition of 300 ml H₂0 + 300 ml CHCl₃. The mixture was vortexed and then centrifuged to resolve the phases. The non-aqueous phase was collected and spotted on thin-layer chromatography (TLC) plates for analysis.[5]

 

Chromatography and analyses:

TLC to characterize the carotenoids was performed on silica (60⁰F 254) aluminum sheets (Merck, Darmstadt, Germany) developed with butanol 5% (v/v), hexane 76% (v/v), ethyl acetate 9.5% (v/v), and dichloromethane 9.5% (v/v). American Spice Trade Association (ASTA) measurements were performed as described by Wall and Bosland (1998).

 

Method -6:

Materials:

Fresh Capsicum fruit (dissected into pericarp tissue, frozen at -80 °C or lyophilized pericarp tissue).

 

Reagents:

N₂ gas, CHCl₃ (HPLC grade), 2-propanol, Methanol, KOH, Methyl-t-butylether, β-carotene, Lutein, Lycopene, Antheraxanthin, Capsanthin, Capsorubin, Zeaxanthin, Violaxanthin, β-cryptoxanthin, MethanolicKOH.

 

Instruments:

Polytron generator Temperature block HPLC system equipped with a photodiode array detector and YMC carotenoid column, Centrifuge, Microcentrifuge, UV/ Visible spectrophotometer, Vortex mixer, Fume chamber, Nitrogen evaporator.

 

Method:

Extraction Procedure:

1.     Using dried pericarp samples use 0.25 to 0.5 g for each extraction, grind samples to a powder in a mortar and pestle. Chop the frozen pericarp into small pieces using a manual food chopper.

2.     Place samples in a 50 ml plastic tube, add 25 ml CHCl₃ and use the polytron to thoroughly homogenize the sample, 20 to 30 sec.

3.     Allow the homogenized tissue to sit in the CHCl₃ for 30 min with occasional mixing by either a vortex mixer.

4.     Filter the CHCl₃ extract using Whatman 1 filter paper and a vacuum filter.

5.     Transfer filtrate to a 40 ml glass amber vial and evaporate the solvent using a stream of N₂ gas and gentle heating (50 °C). A nitrogen evaporator works well for this step.

6.     Resuspend the dried sample in 1.1 ml of 2-propanol by using sonication.

7.     Material can be stored at -20 or -80 °C for several weeks.

8.     We keep the extracted sample out of bright light as much as possible. During the time the sample is on the nitrogen evaporator in the fume chamber, we keep the lights off in the fume chamber; we store the samples in amber vials and we keep the vials in the dark as much as possible.

TLC (General Procedure):

The stationary phase (Silica Gel - G) is applied onto the plate uniformly and then allowed to dry and stabilize.

1.     With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.

2.     Then, samples solutions are applied on the spots marked on the line in equal distances.

3.     The mobile phase is poured into the TLC chamber to a leveled one centimeters above the chamber bottom. A moistened filter paper in the mobile phase is placed on the inner wall of the chamber to maintain equal humidity.

4.     Now, the plate prepared with sample spotting is placed in the TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.

5.     The plate is then immersed, such that the sample spots are well above the level of mobile phase for development.

6.     Allow sufficient time for the development of spots. Then remove the plates and allow them to dry. The sample spots can now be seen in a suitable UV light chamber or any other methods as recommended for the said sample.

 

Observation for TLC:

Method-1: Mobile Phase - petroleum ether (90%), ethyl acetate: ethanol (2:1), n- hexane.

Method-2: Mobile Phase- Butanol 5% (v/v), hexane 76% (v/v), ethyl acetate 9.5% (v/v), and dichloromethane 9.5% (v/v).

Method-3: Mobile Phase - petroleum ether (90%), ethyl acetate: ethanol (2:1), n- hexane.

Method-4: Mobile Phase - petroleum ether (90%), ethyl acetate: ethanol (2:1), n- hexane.

Method-5: Mobile Phase - Butanol 5% (v/v), hexane 76% (v/v), ethyl acetate 9.5% (v/v), and dichloromethane 9.5% (v/v).

Method-6: Mobile Phase-  Butanol 5% (v/v), hexane 76% (v/v), ethyl acetate 9.5% (v/v), and dichloromethane 9.5% (v/v).

 

Methods of extraction for Annuum Capsicum red dye	Practical yield obtained by using different method	The Observed Rf. value of TLC for each methods
Method-1	2.88%	0.56
Method-2	2.18%	0.41
Method-3	1.16%	0.48
Method-4	3.20%	0.31
Method-5	2.44%	0.56
Method-6	2.42%	0.35

 

Extra information:

Uses: Capsicum is useful in many ways in many conditions as follows:

1.     As a spice: the sweetvariety is called as bell peppers and the hot ones as chillies.

2.     In GI disorders: Intestinal gas, upset stomach, cramps, stomach pain, diarrhea, etc.

3.     In cardiovascular disorders: Prevents heart disorders and improves blood circulation and helps decrease blood cholesterol.

4.     In skin disorders: Due to its counter irritant property it is used in the form of ointments, plasters etc to treat rheumatism, shingles, lumbago, etc.

5.     In neuronal disorders: It is used to relieve nerve pain associated with diabetes, HIV, fibromyalgia and back pain.

6.     As an analgesic: It is used to reduce pain associated with osteoarthritis, rheumatoid arthritis, migraine, sinusitis, etc.

7.     In self defense sprays- Due to the unbearable burning sensation when in contact with mucous membranes it is used to spray in the eyes to protect one self. It can cause intense irritation and immediate blindness for at least 30 mins with no permanent damage

8.     Color is one of the crucial factors for the consumer’s acceptability of any processed foods.

 

CONCLUSION:

The study carried out is significant because organic dyeing helps to preserve the traditional art of dyeing and also provides employment and yields economical and ecological benefits. The process of extraction and dyeing is environmental friendly and causes minimum environmental or atmospheric pollution. Use of other mordants may also be considered forim proving the fastness of dyed cloth further research will help to explore the important properties of dye extracted from chili skin.

 

REFERENCES:

1.      Dan Chen and Zanmin, Study on Extraction and Purification Process of Capsicum Red Pigment, Journal of Agriculture science, Vol.1, No.02, December 2008.

2.      Kulkarni S. S., Bodake U. M., Pathade G. R., Extraction of Natural Dye from Chili (Capsicum Annum) for Textile Coloration, Universal Journal of Environmental Research and Technology2011 Vol 1 58-63.

3.      Vinoth Kumar and Rama Prabha, Extraction and analysis of natural dye, Scholars Research Library J. Nat. Prod. Plant Resour, 2018, 8 (2): 32-38.

4.      Irena Perucka a, WiesawOleszek, Extraction and determination of capsaicinoids in fruit of hot pepper Capsicum annuum L. by spectrophotometry and high-performance liquid chromatography, Food Chemistry 71 (2000) 287-291 Analytical, Nutritional and Clinical Methods Section.

5.      Richard D. Richins, Laura Hernandez, Barry Dungan, Shane Hambly, F. Omar Holguin, and Mary A. O’Connell, A ‘‘Green’’ Extraction Protocol to Recover Red Pigments from Hot Capsicum Fruit, Hortscience 45(7):1084–1087, 2010.

6.      Richard D. Richins, James Kilcrease, Laura Rodgriguez-Uribe, Mary A. O’Connell and Edited by Arsalan Daudi, Carotenoid Extraction and Quantification from Capsicum annuum, Bio-protocol, Vol 4, Iss 19, October 05, 2014.

 

Received on 07.10.2019       Modified on 31.10.2019 Accepted on 10.11.2019      ©A&V Publications All right reserved Research J. Science and Tech. 2019; 11(4):281-286. DOI: 10.5958/2349-2988.2019.00040.8