INTRODUCTION:

Turmeric is a flowering plant of the ginger family, Zingiberaceae, the roots of which are used in cooking. The bio-active polyphenol component of turmeric is curcumin, also known as diferuloylmethane (C21H20O6), with an ability to prevent and cure diseases. Turmeric contains about 2-5% curcumin alone.

Commercial curcumin contains three main types of curcuminoids, i.e., curcumin (diferuloylmethane or “Curcumin I" about 77%), demethoxy curcumin ("Curcumin II" ∼17%) and bis demethoxy curcumin ("Curcumin III" ∼3%) 5). Curcumin (diferuloylmethane renders its bright yellow colour to turmeric as mentioned in Figure 1. Curcuminoids exhibit a broad spectrum of biological and pharmacological activities including anti-oxidant, anti-inflammatory, anti-bacterial, anti-fungal, anti-parasitic, anti-mutagen, anti-cancer and detox properties^1-2 as given in figure 1. It is known to be a good antioxidant, anti-inflammatory agent, anti-carcinogenic agent, anti-mutagenic agent, and anti-coagulant, widely used for combating a range of health issues^3-7.

Curcumin is a liposoluble compound and can be easily dissolved into an organic solvent such as methanol, ethanol, and acetone. However, poor water solubility often limits its biomedical uses using aqueous systems. This observation prompted us to examine turmeric extracts as a delivery system for curcumin and to examine the possibility of turmeric extract itself as candidate agent for pharmacologic evaluation. In this preliminary study, we used different solvents to extract the crude turmeric material and compared the curcumin concentrations in these extracts. The choice of solvents for extraction is restricted to the few solvents of defined purity allowed by national and international food laws in the processing of food materials. Hexane, acetone, alcohol (ethanol, methanol), isopropanol and ethyl acetate are used in the extraction of oleoresins of spices. From consideration of solubility of active constituents, the curcuminoids are poorly soluble in the hydrocarbon solvents. Alcohol and acetone are good extractants and the yields can also be expected to be high because of extraction of non-flavour components.

The most conventional method for extraction of curcumin has been Soxhlet extraction with heating time ranging as long as up to 12 h. But this method was time consuming, laborious and makes use of bulk amount of organic solvents. As the heating process continues for long hours, the approach possibly involves high risk of thermal decomposition of target molecules. Soxhlet extraction method operates through cell permeation followed by solubilizing the active constituents by the extracting solvent. Curcuminoids present inside the oleoresin cells which in turn are covered by tightly packed cork cells probably make the entry route for the solvent and time consuming. Soxhelet extraction of turmeric powder with acetone gave a yield of about 4.1% containing in 3 hours. Acetone as solvent was slightly superior to alcohol and ethyl acetate, the curcuminoids content also is on the high side, suggesting selective extraction. The results of extraction with acetone have, however been reported to give high yields of curcuminoids than alcoholic and remaining extraction. A number of studies are undertaken to separate curcuminoid pigments by thin layer chromatography (TLC), column chromatography. HPLC method was sensitive, precise, and accurate for detection and quantification of curcuminoids in the extract of rhizomes curcuma longa. Curcuminoids are polyphenols of hydrophobic nature with very low water-solubility, low stability, higher metabolism and very low absorption rate. Together, these properties decrease the bioavailability of curcuminoids limiting its clinical use. Nevertheless, drug delivery systems have been modified to enhance the biosorption and permeability of curcuminoids and to decrease its presystemic degradation in order to overcome this limitation^8-14. Phytochemical analysis of the crude turmeric aqueous extract was used to identify key secondary metabolites. Furthermore, antioxidant assays were used to assess the bioactivity of purified curcuminoids.

Figure 1:

Figure 2:

The present study describes screening of extraction method for isolation of curcuminoids from turmeric rhizome using non-polar solvents for extraction, isolation, identification and purification of curcuminoids followed by its analysis by HPLC, UV and TLC^15-16.

MATERIALS AND METHODS:

Conventional extraction using Soxhlet:

The turmeric powder was provided as a gift sample from department of pharmacognosy AGI. The Soxhlet extraction as the reference method was performed as follows: 20g ground turmeric powder was weighed and embedded in a thimble and put in the Soxhlet apparatus which was gradually filled with 150ml of acetone as the extraction solvent¹⁷. The experiment was carried out at 56.63^oC within 6hrs. Acetone was separated using rota apparatus and the product i.e., curcuminoids dried and weighed. The dried powder was further used for analysis.

Ultrasound-assisted extraction of curcuminoids:

An ultra sonic bath (citizen scales) with tank capacity of 0.8 liter was used for extraction of curcuminoids. The bath power was 90W with 37KHZ frequency. For extraction 20g turmeric was dissolved in 150ml acetone and sonicated in ultrasonic bath^18-23. The extraction was performed at 25-30^oC for 30mins.The extract was filtered and dried.

Distillation method for extraction of curcuminoids:

20g ground turmeric powder was weighed and placed in to the distillation apparatus which was gradually filled with 150ml acetone as the extraction solvent²⁴. The experiment was carried at 56.63^oC within 30min. The product i.e., curcuminoids obtained upon drying was weighed.

Characterization of curcuminoids extracts:

High performance liquid chromatography (HPLC):

HPLC analysis of individual curcuminoids was performed according to Siregar et al. (2017) using Shimadzu LC-20AD (Kyoto, Japan) equipped with Rheodyne 7725i injection valve with a 20μL loop volume and binary gradient pump. Detection was carried out with Shimadzu Photodiode Array Detector (SPD-M20A) operated at a wavelength of 425nm. Chromatographic separation was performed using Waters X-Bridge C-18 (250mm × 4.6mm i.d; 5μm), set at 45°C. The mobile phase used consisted of a binary mixture of acetonitrile- acetic acid 3.00% (49:51 v/v), i.e., delivered in an isocratic manner with flow rate arranged at 1.08mL/min. For the preparation of a stock solution of samples, an accurately weighed amount of samples (about 200.0mg) was transferred into a 25mL volumetric flask, diluted with 10mL methanol, sonicated for 30min, and then diluted with mobile phase to volume. The samples were homogenized and centrifuged for 10min at 10,000rpm. A portion of sample stock solution was diluted (1 in 20mL for CUR and 2.5 in 5mL for DMCUR) with mobile phase, mixed, and then filtered using 0.45μm filters before being injected into HPLC system^25-26.

Quantification of extracted curcumin was performed using the peak area at specific retention times and standard curve. The amount of extracted curcumin (% w/w) from turmeric was calculated as follows:

M_{extracted
curcumin}

Curcumin yield (%) = ---------------------- x 100

M_{pristine
turmeric}

Thin layer chromatography (TLC) of the curcuminiods:

A glass plate layered with 2mm thickness of silica gel and activated at 105^◦C for 15min after drying. Ethanolic solution of curcuminiods extract was spotted on this plate. Then the plate was run in a chromatographic beaker containing chloroform: Ethanol: Glacial acetic acid (9.4:0.5:0.1) as mobile phase²⁷.

Analysis of curcuminoids by UV-Vis Spectroscopy:

The extracted solutions were also analyzed for the presence of curcuminoids by UV-Vis spectrophotometer (Shimazdu). An accurately weighed quantity of curcumin extracted samples of 10mg was dissolved in 10ml of ethanol which gave a solution of 10000 ppi then from the prepared solution sample dilutions were made to prepare 100, 200, 300 ppi solutions and checked for wavelength absorption in an amber colour volumetric flask. 1ml of the above solution was diluted with ethanol to 10ml in amber colour volumetric flask and the resulting solution was scanned for UV-Vis absorption using UV-Vis spectrophotometer in the range of 400nm to 600nm and λ-max was determined. Furthermore the spectrum of curcuminoids (λ-max) was compared with reported λ-max of the reference spectrum of curcuminoids^28-29.

RESULTS:

Various extraction protocols for the separation of curcuminoids from turmeric were investigated. Even though the Soxhlet extraction was considered as the base method and the extraction yields were compared. Soxhlet extraction is one of the foremost and most common extraction techniques where in long extraction time at high temperature facilitate the extraction of target compound; moreover, the repeated contact of solvent with turmeric can enhance the extraction yield. The results of yield were confirmed by analytical techniques.^30-32

Extraction of curcumin from turmeric was carried out using ultrasound waves and the effect of sonication time (30 min) and temperature (20-30°C) on curcumin extraction yield was examined. The extraction of curcuminoids as ultrasonic temperature increased from 20 to 30°C increased, but increase of temperature beyond 30°C decreased the yields considerably because of evaporation of acetone which was used as extraction solvent or partial destruction of curcumin during sonication. The results of yield were confirmed by analytical techniques.^33-37

Extraction of curcuminoids from turmeric was carried out using distillation of at 56.6^.c for 30 minutes wasindicated in the Figure 3 (a), (b), (c).

HPLC Profile of the extracted samples:

Distillation product 0.1mg:

Figure 3 (a): Soxhlet product 0.1mg

Figure 3 (b): Sonicator product 0.1mg

Figure 3 (c)

Figure 4: UV spectra of sonicated extract (a), (b), (c)

To confirm the presence of curcuminoids in the extracted sample, UV-Vis spectroscopy analysis was conducted.^38-39 The absorbance spectra of standard and extracted curcuminoids using different methods were collected in the range of 300 to 600nm as shown in Fig.4 The standard curcuminoids represented an absorption peak at around 425nm as shown in Figure 4.

Thin layer Chromatography of extracted curcuminoids:

Sample was run on TLC plate; three different spots of curcumin were obtained. After drying sonicated extract were weighed and weight percentage of curcumin, de-methoxycurcumin, and bis-demethoxy curcumin, RF values for these were calculated and found to be similar that of reported values as indicated in Figure 5 and Table 1.

Standard sample:

Sonicated extracted sample:

Figure 5: TLC for standard and sonicated extracted sample of curcuminoids

DISCUSSION:

The better yields of curcuminoids were observed at the said temperature and time. The results of yield were confirmed by analytical techniques. The HPLC profile of the Curcumin, Demethoxycurcumin, and Bisdemethoxycurcumin shows single peak at the retention time of 3.213, 2.93, and 2.85 min respectively indicated in the Figure3 (a), (b), (c).

In UV analysis the characteristic peak which corresponds to the diarylheptanoid chromophore group of curcumin was observed in the spectrum of different samples obtained from Soxhlet, distilled, ultrasound-assisted extraction. Also, the intensity of the peaks corresponds to the efficiency of the implemented extraction methods where Soxhlet extraction was the most effective one followed by sonicated extraction, distilled extractions showed almost same performance. Sonicated extract, Soxhlet extract, distilled extract were analysed on spectrophotometer at 425nm with respect to standard graph. After concentrating each extract total yield was determined and percentage yield of curcuminoids were analysed by spectrophotometer at 425nm as shown in Figure 4. From the above three extracts it was proved that sonicated method of extraction had given good yield. The absorbance of test solution measured at 425nm and the percentage of curcuminoids was calculated by calibration curve method. The method was validated for several parameters like linearity, accuracy. Calibration curve of curcuminoids was then plotted with absorbance on y-axis and curcuminoids concentration on x-axis.

The TLC of the standard and sonicated samples were analyzed by running standard curcuminiods along with the sample which had shown the values of sonicated extract were near to the standard as given in the Table 1. The images Figure 5 illustrated the separation of curcuminoids of standard and sonicated using TLC and RF Values indicated the similarity of the sonicated sample to the standard.

ACKNOWLEDGEMENT:

Authors are thankful to Dr. P. Rajeshwar Reddy, Chairman and Dr. Vasudha Bakshi, Dean, Anurag group of Institutions (Formerly Lalitha College of Pharmacy), Venkatapur, Ghatkesar for providing the facilities for completion of this research work.

CONCLUSION:

Curcuminoids were extracted from turmeric through 3 methods-distillation, sonication and soxhelation. Obtained curcuminoids was further analysed by bio-analytical techniques. The purpose of this work was to evaluate the efficiency of different extraction methods for extraction of curcumin from turmeric as compared to the Soxhlet extraction as the most common and traditional extraction method. The extraction yields obtained by Soxhlet extraction were considerably higher than those obtained from other methods; however, the harsh operation condition of this method including long extraction time (6 h) and high extraction temperature (56.6°C) makes this method unattractive. While the subsequent extraction methods employed in this study resulted in better yields and seem to be more promising from both economic (much less time and energy consumption compared to Soxhlet) and environmental friendly (consumption of much less solvent). These were evaluated for higher yield of curcuminoids through HPLC. HPLC result of extraction of sample from the three methods revealed that extract obtained from sonicated method had higher yield as compared with IP standard. In comparison to all methods of extraction sonication proved to be the best. Separation of curcuminoids from turmeric using number of advance methods was inspected and the outcomes were compared to those obtained from Soxhlet as the most common and reference method. Among these modern extraction methods ultrasound-assisted extraction show high extraction yields as high as Soxhlet method. Even the applications such as low extraction temperature, short extraction time and use of very slight amount of solvent makes it more favourable extraction methods in comparison with other extraction methods.

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Received on 15.07.2020 Modified on 17.10.2020

Research J. Pharm. and Tech. 2021; 14(9):4615-4620.

DOI: 10.52711/0974-360X.2021.00802

Sr No.	TLC of mobile phase	Ratio	RF Values
			Soxhlet			Sonication			Distillation
			C	DMC	BDMC	C	DMC	BDMC	C	DMC	BDMC
1	Chloroform: ethanol: Glacial acetic acid	9.4:0.5:0.1	0.6	0.59	0.55	0.7	0.57	0.5	0.6	0.57	0.52
2	Chloroform: ethanol: Glacial acetic acid [reference]	9.4:0.5:0.1	0.65	0.58	0.54	0.75	0.56	0.3	0.62	0.55	0.5