CHAPTERS
Chapter 3: Oil extraction and physico – rheological characterization of organic extracts obtained from the seeds of A. nigra
3.2 Materials and methods
3.2.1 Collection of fruits of A. nigra
The fruits of A. nigra grow in 3 stages viz. immature (green), partially mature (red) and mature (black). Sufficient quantity of healthy fruits of each stage were collected in an autoclave bag during June - August and from November – January every year between 2014 – 2016 from the wildly growing plants in and around the Indian Institute of Technology Guwahati (IITG) campus (26◦12.476’ N to 91◦41.965’E). They were shade dried and stored at room temperature (25°C) till further processing.
3.2.2 Processing of dried fruits
The ectocarp and the pericarp portions of the dried fruits were removed manually and the seeds from the trilocular chambers of the fruits were collected and stored in an air tight bag at 25°C until further usage.
3.2.3 Preparation of organic extracts
Various extraction methods viz. conventional maceration, soxhlet extraction, microwave assisted extraction, ultrasound assisted extraction and many other methods are available for the extraction of chemicals components from plant materials in the form of crude extracts. In the current study, 2 different methods were used for preparation of crude extracts from the seeds of A. nigra.
3.2.3.1 Soxhlet extraction
Among all the methods, hot solvent extraction using soxhlet extractor is reported to give significantly higher yield in terms of phenolic content and other secondary metabolites in terms of economic investment (Aspé and Fernández 2011). Seeds from the 3 different stages of fruits were washed, cleaned and shade dried for a few days. The seeds were then powdered and subjected to hot solvent extraction in soxhlet apparatus using organic solvents (n-hexane, ethyl acetate, methanol), with increasing polarity. After each extraction, the same samples were dried properly to remove any traces of previous solvent so as to reduce the carry over effect and subjected to the next polar solvent. The parameters of each extraction were carefully recorded. The extract obtained, was concentrated by rotary evaporator (Cole parmer, USA), and stored for further analysis.
All the solvents were of HPLC grade (Ranken, India).
Oil extraction and physico – rheological characterization….
3.2.3.2 Room temperature extraction
The dried seeds were macerated into a fine powder using a mortar and pestle, with continuous addition of organic solvent (n-hexane and ethyl acetate), with increasing polarity. After maceration, the mixture was sealed tightly and kept overnight at 25ºC. The extract obtained was filtered and then, concentrated using rotary evaporator. The seed-to- solvent ratio was optimized as per the parameters given in Table 3.1.
Table 3. 1Parameters for optimization of seed-to-solvent ratio
Sr. No. Seed-to-solvent ratio (w/v) Seeds (g) Solvent (ml)
1. 1:4
32.05
128.2
2. 1:6 192.3
3. 1:9 288.45
3.2.4 Physico – rheological characterization 3.2.4.1 Physical properties
Color, odor and state of the extracts were noted at room temperature by visual inspection (Onyeike and Acheru 2002). Determination of refractive index was done using refractometer (Atago DR-A1, India). Density was calculated by measuring the weight of known volume of samples on a balance (Sartorius; max. 220 g ± 0.1 mg) and then changing the calculation units.
3.2.4.2 Fourier transform infrared (FTIR) spectroscopy
A drop of each sample was cast on a small amount of potassium bromide (KBr; HiMedia, India) and made into a pellet in preparation for FTIR analysis. The spectrum was recorded in the spectrophotometer (Perkin Elmer, Germany) in the range of 700-4000 cm-
1.
3.2.4.3 Contact angle
Water contact angle was measured using a drop shape analyzer (Kruss, Germany), having a needle diameter of 0.5 mm, droplet size 4 µm, droplet rate of 0.16 per min and having a frame rate of 60. Samples were spread over a glass slide and the contact angle was measured.
Oil extraction and physico – rheological characterization….
3.2.4.4 Optical rotation
Optical polarimeter (AUTOPOL II, Rudolph Research Laboratory, USA) was used to calculate the specific rotation and optical purity of the test samples, measured at a wavelength of 589 nm.
3.2.4.5 Differential scanning calorimetry (DSC)
DSC was performed on a Netzsch DSC instrument. The analysis was completed in two thermal scans; the first cycle was from -30 to 240ºC at a heating rate of 5ºC per min followed by 2 mins of isothermal condition at 240ºC and then cooling from 200 to 30ºC with a cooling rate of 5ºC per min.
3.2.4.6 Thermogravimetric analysis (TGA)
TGA was executed by thermogravimetric analyzer (TGA-4000, Perkin Elmer, USA) under a nitrogen atmosphere with a flow rate of 50 ml/min. The test samples were placed in a alumina crucible and the analyses were performed at a temperature range of 30 to 650ºC at a heating rate of 10ºC per min.
3.2.4.7 Viscosity
Ubbelohde type capillary viscometer with a temperature controlled system was used for the measurement of viscosity of the 2 plant extracts. The concentrations of the samples used for measurement were 0.0001 g/ml, 0.0002 g/ml, 0.0008 g/ml, 0.0014 g/ml and 0.0018 g/ml in chloroform. The measurements were taken at 15ºC, 25ºC and 50ºC to see the effect of temperature on the intrinsic viscosity of the samples. The relative viscosity (Ƞr) of the samples was calculated from the flow time of the pure solvent and the sample solution and the specific viscosity (Ƞsp) was calculated by subtracting 1 from the relative viscosity obtained as shown in equation (1) and (2) respectively.
Ƞr = ………..(1)
Ƞsp = 1- Ƞr………..(2)
where, t = flow time of samples in secs to = flow time of pure solvent in secs
Oil extraction and physico – rheological characterization….
3.2.5 Statistical analysis
All the experiments were set up in a completely randomized manner, performed in triplicate and mean of the three was used for the plots. All graphs were plotted using Origin 8.5; R2 > 0.97.