Extraction and Characterization of Sesbania cannabina (Retz.) Pers. (Dhaincha) Seed Oil for Potential Engineering Applications

Sesbania cannabina (Retz.) Pers. (Dhaincha) is a member of family Fabaceae spread over several countries in tropical and subtropical regions of the world. Sesbania aculeata, Sesbania drummondii, Sesbania grandiflora, Sesbania rostrata, Sesbania sesban, and Sesbania speciosa are other members of this family. The agricultural, nutritional and pharmaceutical applications of Sesbania species are known to farmers, villagers, and the tribes since ages and are well studied by researchers. However, the significance of Sesbania as an industrial crop has not been recognized till now. The objective of this study was extraction and characterization of Sesbania cannabina seed oil (SCSO) for potential engineering applications. The seed oil was extracted with hexane in a Soxhlet extractor. Yield was only 2.32% w/w due to long storage at high temperature in seed house. Sesbania cannabina seed oil methyl ester (SCSOME) was prepared via esterification and transesterification for analysis of fatty acid composition of extracted oil. SCSO has high iodine value (118 g I2/100 g) and high saponification value (185.79 mg KOH/g) making the oil suitable for use as candle stocks or in soap making. However, these applications were ruled out on account of being insignificant for oil available in limited quantity. The oil has high viscosity index (174.19), high onset (382°C) and offset (450°C) decomposition temperatures, endothermic nature, and shear rate thickening behaviour. These properties make SCSO a good candidate for application as specialty lubricant required under severe operating conditions of high temperature and high shear rate or as insulating and cooling transformer oil.

The agricultural, nutritional and pharmaceutical applications of Sesbania species are known to farmers, rural people and tribal communities since ages particularly in Indian subcontinent. The major application of Sesbania is as a leguminous crop to increase the soil fertility. It is a fast-growing, succulent and easily decomposable green manure crop which on mulching with soil increases the quantity of organic matter and nitrogen in soil 6 . This in turn results in higher yield for wheat, rice, maize, sorghum, sugar cane, etc. 4 . The concurrent growing of Sesbania with rice significantly reduces the count of grass, weeds and sedges in the field resulting in higher yield of rice due to increased nutrient uptake from soil in a competition free environment 7 . The other less important applications of Sesbania species in traditional agroforestry include as cover crop, shade plant, windbreak, live support fence, livestock feed, aquafeed, fibre, wood for light construction work and firewood 2,8,9 . The stems are used as poles in fishing and in fencing around agricultural fields and gardens. The stem wood has been reported to be used for mushroom cultivation in Mekong river delta of southern Vietnam 10 . The fibre obtained from the stem is used in making high strength ropes and fishing nets 11 .
The leaves, flowers, pods and seeds of Sesbania constitute a valuable source of livestock feed. Sesbania leaves contain 30 protein and good amino acid composition and are therefore considered a high quality fodder for livestock with reported increase in milk and meat production 12 . Sesbania seeds contain 29-33 crude protein, 4.7-6 crude lipid, 11-16 crude fibre, and 44-47 carbohydrates 13 . Some tribal sects of South India are reported to consume mature seeds after proper cooking 14,15 .
Sesbania occupies a unique place in traditional Indian medicines for treatment of various health disorders on account of healing properties in different parts of the plant. The roots of Sesbania are used as alexiteric against the effects of poison , anthelmintic to destroy parasitic worms , carminative to prevent formation of gas in the gastrointestinal tract and facilitate its expulsion , collyrium medicated eyewash , contraceptive to prevent pregnancy , diuretic causing increased passing of urine , galactagogue to promote the flow of mother s milk and hepatoprotective to protect damage to the liver . Some other pharmaceutical applications of roots include the treatment of dysuria painful urination , leucoderma loss of skin colour , fever, ulcer, and diabetes 12,16,17 . Similarly, barks are effective in treatment of anaemia, bronchitis, dysentery, inflammations, leucorrhoea, liver cirrhosis, tumour and hypertension 12 .
The extract of Sesbania leaves in the form of tea is believed to contain anthelmintic, antibiotic, antispasmodic, antitumor, contraceptive 14,18 and sedative properties. Leaf paste is often used in seeking relief from skin diseases and inflammatory rheumatism 12 . Leaves wash is used as prevention to tsetse flies and is also given to infants against a whooping cough 17 . Sesbania leaves have good concentration of antidiabetic -pinitol or O-methyl inositol l-methoxy-2,3,4,5,6-penta hydroxy cyclohexane 6 . Pinitol is also active against larval growth of major agricultural pest corn earworm moth Helicoverpa zea 19 . Sesbania flowers are used as poultice to cure skin afflictions 15,16 , whereas the flowers smoke is an effective mosquito and insect repellent 17 .
Sesbania seeds are good astringent causes skin or other tissues to tighten , emmenagogue promotes menstrual discharge and stimulant. They are also used in treatment of diarrhoea, fever, spleen enlargement, ulcers and as purgative demulcent 12 . Seed powder is extensively applied on infected area in the treatment of ringworm, skin diseases, wounds 14,16,18,20 and headache 15 . Seed powder is also given to stimulate appetite 17 . The seed meal with about 53 protein is found as a suitable medium for production of antibiotic fungi Penicillium 11 . Seed oil is an effective pain reliever particularly in arthritis 15 .
The fibre obtained from stem wood of annual Sesbania species can be used in paper industry for pulp making 8,20 . Sesbania seed oil is used in the production of paints, varnishes, and printing inks due to its higher viscosity than other vegetable oils 21 . Sesbania seeds are good source of non-toxic polysaccharide galactomannan mannose-to-galactose M/G ratio: 1.8:1 22 that can be used for several applications in biomedical, cosmetics, food, pharmaceutical, paper and textile industries. In food industry the main applications of galactomannan are as stabilizer, thickener, emulsifier and firming agent in bakery mixes, baby milk formulations, coffee/tea whiteners, dairy and dietary products, fruit-based water gels, ice creams, powdered products, salad dressings, sauces and soups, canned meats and frozen and cured meat products 23 . In pharmaceutical industry galactomannan is used in manufacturing of tablets to improve their hardness and delivery to the large intestine without release into the upper gastrointestinal tract 15 .
The use of Sesbania in agriculture as a leguminous green manure crop is so popular and established that it has overshadowed the horizon for other applications. For instance, the nutritional and pharmaceutical applications are confined to a few rural and tribal communities without much commercial value. Sesbania seed galactomannan is equivalent to guar gum and is less expensive 15 , howbeit it has not received as much acceptance as guar gum in food industry. Several research groups in India 24 , Pakistan 25 , Bangladesh 26,27 and Vietnam 10 have confirmed the potential of Sesbania fibre for pulp making, but it is hardly used as raw material in pulp and paper industry as compared to eucalyptus and poplar. Authors of this article observe that the importance of Sesbania as an industrial crop is not yet recognized and corresponding efforts in this direction are lacking. It would be difficult for our small research group to conduct research on all aspects of Sesbania with equal authority; henceforth we fix our attention on Sesbania cannabina seed oil SCSO for applications other than agricultural, nutritional and pharmaceutical. The main objective set for this study was to extract oil from Sesbania cannabina retz. pers. Dhaincha seeds with hexane by hot extraction in a Soxhlet extractor and its characteriza-tion for potential engineering applications. This study might seek the attention of researchers towards this species for further studies culminating in increased commercial utilization.

Materials
Sesbania cannabina Dhaincha seeds were purchased from local seed store in vicinity of Kanpur University. Hexane LR and Potassium hydroxide pellets LR were purchased from Avantor Performance Materials India Limited, Ankleshwar. Methanol GR and anhydrous Sodium sulphate were purchased from Merck Specialities Pvt. Limited, Mumbai. Sulphuric acid abt. 98 LR was purchased from S. d. Fine-chem Limited, Mumbai. Silicon high vacuum grease was purchased from Loba Chemie Pvt. Limited, Mumbai. Phenolphthalein indicator solution 1 was purchased from Central Drug House P Limited, New Delhi. All chemicals were used as obtained from vendor without any purification.

Plant Description
Sesbania cannabina Fig. 1 is an annual erect plant that can grow up to a height of 3.5 m. The stem is green in colour that becomes somewhat woody and is much branched on the upper side. The mature leaves are 5-20 cm long and are in 12-30 pairs of leaflets. Each leaflet is 5-20 mm long and 2-4 mm wide with rounded leaflet tip. The raceme has 1-5 pea-like flowers which are yellow to yellowish orange with dark purple streaks on the corolla.
The pods are near cylindrical, straight or little bent 12-20 cm long and 2.5-4 mm in diameter containing 20-35 seeds. The seeds are smooth dark green cylindrical and 3-4 mm long. The plant is widely distributed over several countries of the world. A partial list includes India, Nepal, Bangladesh, Pakistan, Myanmar, Malaysia, Mauritius, Indonesia, Vietnam, Philippines, Ghana, Southern China, Australia, New Guinea, New Caledonia and South Pacific Islands 28, 29 .

Experimental Setup
The extraction of SCSO was carried out in a 60 ml capacity Soxhlet extractor. The extractor capacity is the volume of the solvent held in the extractor to the cusp of the siphon tube. The various components of experimental setup are shown in Fig. 2. The inner joint at the bottom of the extractor was connected to the centre neck of a three-  necked round bottom boiling flask. The outer joint at the top of the extractor was connected to the reflux condenser. The condenser refluxes the solvent vapours back to the extractor to prevent any solvent loss. The condenser is also useful in sustaining the atmospheric pressure inside the boiling flask 30 . An oil-filled thermowell was fitted in one side neck of boiling flask to put the thermal sensor for continuous temperature measurement. The other side neck was used as sampling port. REMI 2RML heater cum magnetic stirrer was used to provide necessary heat and stirring to feed material. PTFE coated magnetic stirrer bar was used in place of top mounted mechanical stirrer. A highly efficient custom-made PID controller manufactured by Blue Bell Industries, Kanpur was used to maintain the temperature in the boiling flask within 0.1 of the set values. An external tank equipped with a small capacity centrifugal pump supplied cold water to reflux condenser. Running tap water was not used in the study to prevent wastage of this valuable natural resource. The synthesis of Sesbania cannabina seed oil methyl ester SCSOME was carried out in the same experimental setup except that the Soxhlet extractor was displaced from the assembly. Stainless steel S.S. sample holders were specifically designed for oil extraction. Three cylindrical sample holders, each of dimension 180 mm 28 mm were made from 1 mm thick perforated S.S. sheet. The sample holders were then wrapped with multiple layered muslin cloth to prevent slippage of fine seed particles during extraction. These sample holders are highly economical, durable and are convenient to work with and easy to clean than conventional cellulose or glass fibre thimbles.

Oil Extraction
The seeds were manually cleaned to remove dust, dirt and other foreign particles chiefly the wheat and rice grains and seasonal pulses. The cleaned and dried seeds were grinded in a domestic grinder to produce powdered seed particles. Around 60 g of powdered seeds were extracted with 0.5 L of hexane in Soxhlet extractor at 68 for 8 hours. Hexane condensation rate was fixed at 150 drops/min for the entire duration 3 . After extraction the hexane was recovered from the oil by blank extraction. In blank extraction the experiment was run as usual but with empty extractor having no seeds. In this manner the pure solvent was collected in extractor and withdrawn carefully before running off the siphon. The residual traces of hexane were removed by heating the oil in water bath at 50 31 . The extracted oil was then dried over anhydrous sodium sulphate 32 and stored at laboratory temperature in closed glass bottle. The average oil yield was merely 2.32 w/w that necessitated several batches to run in order to collect sufficient quantity of oil for characterization purpose. The oil extraction data for some experimental runs are given in Table 1.

Physicochemical analysis
The density, acid value, free fatty acid, saponification value, iodine value, peroxide value, unsaponifiable matter, moisture content, colour, and refractive index of Sesbania cannabina oil were determined following Indian Standard Methods of Sampling and Test for Oils and Fats Part I Sampling, Physical and Chemical Tests Revised IS: 548 Part I -1964. The viscosity index VI value was determined on the basis of known viscosities of oil at 40 and 100 using ASTM D2270 standard method. The higher heating value HHV also called gross calorific value of the oil was determined non-calorimetrically using equation 1 involving saponification value and iodine value of the oil 33 .
HHV 49.43 0.041 SV 0.015 IV 1 2.5.2 Methyl ester preparation for GC-MS SCSOME was synthesized via two stage esterification and transesterification protocol on account of acid value of SCSO exceeding the threshold limit of 2 mg KOH/g oil. First the acid-catalyzed esterification of SCSO was carried out with sulphuric acid as catalyst. After successful completion of first stage reaction, base-catalyzed transesterification was initiated with potassium hydroxide as catalyst. At the end of the process a clear emerald green liquid methyl ester was formed. A detailed description of two stage process for vegetable oil methyl ester formation can be found elsewhere 30 .

GC-MS analysis
The fatty acid profile of SCSOME was determined using an Agilent gas chromatograph GC-FID Model 6890, inte-

Viscosity analysis
The viscosity of SCSO was measured using TA instruments Discovery Hybrid Rheometer model HR-3 Peltier plate and cone geometry diameter 60 mm, angle 1/2 degree under air at varying shear rates.

Results
SCSO characteristics were determined following Indian standard IS: 548 Part I -1964 methods and the results are summarized in Table 2. The acid value of oil indicates its degree of purity, aging and quality upon storage 21 . The iodine value is an indication of the degree of unsaturation DU or the average number of C-C double bonds in its constituent fatty glycerides. The saponification value is an assessment of average molecular weight or carbon chain length of all the fatty acids present in test sample as triglycerides. Higher saponification value corresponds to smaller average molecular weight and the smaller average fatty acid carbon chain length means higher percentage of short carbon chain fatty acids of triglycerides and vice versa 36 . Peroxide value is a measure of lipid oxidation and determines the oil quality. Freshly refined oils have peroxide value less than 0.05 37 . VI is a dimensionless scale used to determine the dependence of viscosity on temperature 38 . High VI corresponds to a small change in viscosity over a large temperature range 39 and the other way around for low VI 40 .
On the basis of GC-MS analysis of SCSOME, the percentage compositions of component fatty acids were calculated and are given in Table 3. SCSO is found to contain 41.36 saturated fatty acids, 51.69 monounsaturated fatty acids and 6.95 polyunsaturated fatty acids. The seed oil is rich in oleic acid 41.34 along with moderate proportions of palmitic acid 13.85 , stearic acid 8.65 and lauric acid 6.35 acid. SCSO contains 1.44 omega-3 alpha linolenic acid which is greater than several commercial oils such as safflower 0.15 , sunflower 0.16 , rice bran 0.45 , and wheat germ and rapeseed 1.2 each 41 . The oil is also a rich source of omega-6 linoleic acid 1.89 . The DU for SCSO was estimated using the following equation 42 : Degree of unsaturation DU monounsaturated fatty acid C n :1 wt.
2 polyunsaturated fatty acid C n :2 wt. 3 polyunsaturated fatty acid C n :3 wt. . DU was calculated to be 70.65 wt. . Density, viscosity and HHV are correlated with the DU. Researchers have determined that the density of vegetable oils increases with reducing chain length and increasing DU; on the contrary, the viscosity increases with chain length and decreases as the DU increases. The HHV of vegetable oils increases with the DU 43 .
TGA-DSC thermograms of SCSO are shown in Fig. 3. It is evident from the TGA plot that thermal decomposition of SCSO took place in a single stage. As the temperature was increased from 50 to 100 , a loss of 0.4616 in weight was observed indicating the presence of small moisture in the oil. The sample displayed remarkable thermal stability by retaining 97 of its initial weight until 223 , 95 until 258 , and 90 until 313 . At 400 , the weight loss was about 40 . This result is attributed to the flash point of oil under study. The onset T onset and offset decomposition T offset temperatures were analyzed graphically from the thermogram at 382 and 450 , respectively, where a weight loss of 63 occurred over a thermal interval of 68 . The maximum decomposition temperature was observed at 409.583 with 0.1592 mg weight loss. The temperature plateau starting at 488.93 showed no further significant weight loss indicating the fire point of oil sample. The weight loss profile for SCSO is given in Table  4.
The results of viscosity analysis are plotted as shear rate Table 3 Fatty acid composition of SCSO.  Fig. 4. The plot shows a rise in viscosity of SCSO with increase in shear rate, which is indicative of shear rate thickening behaviour for test oil.

Discussion
Low oil content in Sesbania cannabina seeds and high free fatty acid in crude oil is attributed to longer storage time and higher temperature at seed storage facility 44 . High iodine value 118 g I 2 /100 g of oil makes it useful for applications such as salad oil, cooking grease, candle stock, etc, whereas its high saponification value 185.79 mg KOH/ g makes the oil useful for soap making industries 36 . High VI 174.19 of SCSO favours its application preferably as a quality lubricant for high temperature operating conditions.
SCSO is rich in omega-3 alpha linolenic acid 1.44 and omega-6 linoleic acid 1.89 , both of which are considered essential to human body as they are important structural components of cell membranes, but are not synthesized by human body. The higher saturated fatty acid content 41.36 of oil is largely responsible for high oxidative and thermal stability in oil leading to a slower deterioration rate of oil characteristics 21 .
TGA observations further confirmed the higher thermal stability of SCSO. The corresponding DSC plot confirms the endothermic nature of SCSO and infers that SCSO can be used in high temperature engineering applications such as insulating fluid in transformers 21 . Such oils are ideal for those applications which involve high speed rotation of machine components, like automobile engines, aircraft engines, power generation turbines, as these oils will retain their lubricity under severe operating conditions 30 .

Conclusion
In this study, SCSO was characterized for applications other than agricultural, nutritional and pharmaceutical. The average crude oil yield extracted from Sesbania can-   nabina seeds was low, whereas the free fatty acid content was high, both due to longer storage time and higher temperature at seed house facility. The study found SCSO to have high iodine value, saponification value, and VI, excellent thermal stability, good endothermic nature, and shear rate thickening property. High iodine value and saponification value make the oil suitable for use as candle stocks or in soap industries. But since the oil content in Sesbania cannabina seeds is very low, use of SCSO for such trivial applications is strongly ruled out. High thermal stability and endothermic nature makes the oil suitable for use as an insulating and cooling liquid in transformers replacing currently applied mineral oils. High thermal and oxidative stability along with high VI and shear rate thickening behaviour of oil indicate towards its potential use as a quality lubricant for severe operating conditions of high temperature, pressure, and shear rate.

Conflicts of Interests
All the authors declare to have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.