Preparation and characterization of renewable bio-polyol from the edible seed oil

An herbaceous plant called chia has opposing, serrated leaves that range in size from 1.5 to 3 inches long and 1 to 2 inches wide. Oval in shape, seeds are roughly 2 mm (0.08 inches) in length and 1 mm (0.04 inches) in width. he lustrous seeds contain darker irregular marks or specks on them and their coat can range in hue from cream to charcoal grey. An annual herb called chia ( Salvia hispanica L .) blooms in the summer. It is about a meter tall and has opposite, petiolate, serrated leaves that range in length from 4 to 8 cm a width of 3 to 5 cm (Ayerza et al., 2010).

herefore, this study concentrates in the obtention of new bio-based polyol from the edible oil extract from the seeds of the Salvia hispanica and also to test the phytochemicals and basic radicals present in the oil extract. In addition, we characterize the prepared polyols using various physical properties and spectroscopic methods. he application of the prepared polyol against certain microbes were also tested.

MATERIALS AND METHODS

OIL EXTRACTION:

he extraction of oil was carried out using AOAC technique Am2-93 (Official Methods and Recommended Practices of the American Oil Chemists’ Society; AOCS Press: Champaign, IL, USA, 1995). In a Soxhlet device, 5g of Salvia hispanica L. seeds were extracted with 100 ml of n-hexane as the extraction solvent. Following an 8-hour period, the n-hexane was eliminated at 40˚C and low pressure. After obtained, the Salvia hispanica L . oil (SHO) was kept in a refrigerator till additional research was conducted.

SYNTHESIS OF POLYOL:

he synthesis of the polyols was done in accordance with the approach outlined by Monteavaro et al. (2005). 5g (5.6 mmol) of oil 9.30 mL (0.162 mol) of glacial acetic acid in 20 mL of toluene, together with a few drops of sulfuric acid, were combined in a three-necked flask that was fitted with an isobaric funnel, reflux condenser, and mechanical stirrer. At room temperature, the mixture was mechanically agitated until it was completely homogenized. Subsequently, 5.30 mL of a 30% H 2 O 2 , solution was added gradually while maintaining the temperature. Following the addition of H 2 O 2 , the mixture was heated for 12 hours to 60˚C. After bringing the reaction mixture down to room temperature, surplus peroxide was removed by stirring it for 20 minutes while adding a 10% (w/v) sodium bisulfide solution. Following that, the mixture was mixed with 50 mL of ethyl ether, and the organic phase was repeatedly washed to a pH of neutral using a 10% (w/v) sodium carbonate solution. In order to extract Salvia hispanica L. (SHP), the organic phase was lastly dried over sodium sulphate and concentrated under vacuum to remove the ethyl ether. CHARACTERISATION OF OIL AND POLYOL: PHYSICO CHEMICAL ANALYSIS:

he acid value of SHP was determined by the volumetric titration method according to AS M D1980-

87. he known quantity of sample is dissolved in neutral butanol-toluene mixture and 3–4 drop of phenolphthalein indicator added, swirling gently and titrated against the 0.1 N alcoholic KOH solution. Equation (1) was used to determine the acid value of SHP.

Acid value= B Х N Х 56.1                          (1)

W where B = Burette reading (ml), N = Normality of alcoholic KOH solution, W = weight of sample (g).

he hydroxyl value of SHP was determined by acetic anhydride-pyridine method according to AS M D4274-16. he calculation of hydroxyl value was done by using Equation (2).

Hydroxyl value = (B - S) × N × 56.1                  (2)

W where B = Burette reading for blank (ml), S = Burette reading for sample (ml), N = Normality of alcoholic KOH solution, W = weight of sample (g).

PHYTOCHEMICAL AND BASIC RADICAL IDENTIFICATION:

Phytochemical screening and basic radical identification were performed to asses the qualitative chemical composition of different samples of crude extracts using commonly employed precipitation and colouration reactions to identify the major and secondary metabolites.

FTIR ANALYSIS:

he chemical structure of the SHO and SHP were identified by F IR on a Bruker A R spectrophotometer.

he spectra were observed in the 600 – 4000 cm-1 wavelength range.

1H NMR SPECTROSCOPY:

he supportive confirmation of chemical structure was given by ¹H nuclear magnetic resonance (NMR)spectroscopy. ¹H spectra of the product were analyzed using Bruker DPX 400 MHz spectrophotometer with CDCl 3 as solvent.

ANTIMICROBIAL ACTIVITY:

Antimicrobial activity was examined using “ he Kirby-Bauer Method” against the number of pathogens including both gram-positive and gram-negative bacteria and also certain fungi. he zone of inhibition of both SHO and SHP were examined against certain pathogens.

RESULTS AND DISCUSSION

PHYSICO CHEMICAL ANALYSIS:

he acid number is expressed as the number of milligrams of KOH required to neutralize the acidity of sample. he OH number is the amount of available reactive hydroxyl groups on polyol molecules. he viscosity was measured using Brookfield viscometer and the density of the polyol were also analysed. he SHP is well dissolved in Chloroform . he results of the certain physico chemical analysis were mentioned in the able 1.

PHYTOCHEMICAL SCREENING:

he preliminary phytochemical study reveals the presence of glycosides, reducing sugars, phenolic compounds and saponins ( able 2) in the SHO. he color change in the extract were also listed in the table. BASIC RADICAL IDENTIFICATION:

Basic radical test shows the presence of bismuth, barium calcium and magnesium in the SHO were listed in the able 3.

FTIR:

he successful conversion of SHO to SHP was confirmed qualitatively by F -IR spectroscopy. Fig. 1 and Fig. 2 shows the F IR spectra of SHO and SHP respectively. he SHP spectra shows a broad band at 3562 cm-1 which were assigned to the presence of a hydroxyl (–OH) stretching vibration. A strong band at 3181 cm-1 was attributed to the presence of aromatic -CH-stretching and bands at 1644 cm-1 and 1400 cm-1 were assigned to the presence of amide band from protein carbonyl stretches and C-O-H bending vibrations. he appearance of new peak at 3562 cm-1 in SHP spectra confirms the formation of polyol.

1-H NMR spectra:

Fig. 3 and Fig. 4 gives the 1-H NMR spectra of the SHO and SHP respectively. he methylene protons of the aliphatic chains may be responsible for the signal seen as a triplet between 1.63 to 1.67 ppm (3H) in Fig. 4. he proton of the hydroxyl group is represented by the sharp singlet peak at 2.731 ppm (6H); the electronegative influence of the oxygen atom is responsible for the absorption shift towards the downfield. he multiplet signal at 3.8 ppm (2H) is indicative of the protons next to the acid’s carbonyl

group. he peak shift downwards as a result of the       present in the range of 5.3 to 5.3 ppm (Fig. 3) in SHO carbonyl group’s electronegative action, which de-shield       vanished, indicating that the SHP structures were the area (Dai et al., 2005). he signals corresponding to       essentially unsaturated. Lastly, the hydroxylation the toluene structure were found at 7.5-7.6ppm as       reaction was verified by the lack of signals in the range multiplet (Fig. 4). his evidence confirmed that the       of 2.8 to 3.3ppm in relation to the epoxide groups (elimination of toluene was ineffective. Furthermore, the       CH(O)CH-). olefinic hydrogen (-CH=CH-) signal that had been Table 1 : Physico chemical analysis of polyol
Parameters	Chia based polyol
Acid number (mg KOH/g)	5.386
OH number (mg KOH/g)	166.54
Density (g/cm3)	0.868
Viscosity (cps)	464
Solubility	Chloroform

Table 2: Phytochemical screening of SHO

EXPERIMENT	OBSERVATION	INFERENCE
NaOH test Extract + 2ml of NaOH	No blue green color obtained	Absence of anthocyanin
Bromine water test: Extract + bromine water	Pale yellow color obtained	Presence of glycosides
1ml of + lead acetate solution	No precipitate obtained	Absence of phenol
5 ml of extract + 2ml of CHCl 3 + 3 ml of conc. H 2 SO 4	No reddish-brown precipitate obtained	Absence of terpenoids
LEAD ACETATE TEST: Extract + 1ml of lead acetate	No red precipitate	Absence of tannins
Extract + CHCl 3 + conc. H 2 SO 4	No purple color obtained	Absence of steroids
Extract + Molisch’s reagent	Purple color is obtained	Presence of reducing sugars
Extract + 2N HCl and remove the aqueous layer and add Mayer’s reagent	No white precipitate obtained	Absence of alkaloids
Alcohol + extract + ferric chloride	Intense color	Presence of phenolic compounds
Extract + water + shake well	Foamy lather is obtained	Presence of saponins
Alcohol + extract + Mg ribbon + Conc. HCl	No color change	Absence of flavonoids
Alcohol + Conc. HNO 3 + NH 3	No reddish orange color is obtained	Absence of xanthoproteins

Table 3: Basic radicals’ identification

EXPERIMENT	OBSERVATIONS	INFERENCE
Extract + KI	No golden spangles	Absence of lead
Extract + NH 4 OH	white precipitate	Presence of bismuth
Extract + cupron reagent + NaOH	No green color obtained	Absence of copper
Extract + potassium ferrocyanide	No white precipitate obtained	Absence of zinc
Extract + dil. HCl + water + H2S	No yellow precipitate	Absence of cadmium
Extract + potassium thiocyanate	No red or blue color	Absence of ferric and cobalt
Extract + dil. HCl + aluminon reagent + (NH 4 ) 2 CO 3	No bright red precipitate is obtained	Absence of aluminum
Extract + conc. HNO 3 + sodium bismuthate + water	No pink color	Absence of manganese
Extract + dimethyl glyoxime + NH 4 OH	No scarlet red precipitate	Absence of nickel
Extract + potassium chromate	Pale yellow precipitate	Presence of barium
Extract + NH 4 OH + ammonium oxalate	White precipitate	Presence of calcium
Extract + Magneson reagent + NaOH	Blue precipitate	Presence of magnesium
Extract +NaOH + Nesseler’s reagent	No reddish-brown precipitate	Absence of ammonium

Table 4: Shows the zone of inhibition against certain microbes

Bacteria	Zone of inhibition
	SHO	SHP	Control (Amikacin)
Proteus mirabilis	12 mm	11 mm	18 mm
Salmonella typi	13 mm	14 mm	19 mm
Fungi	Zone of inhibition
	SHO	SHP	Control (nystatin)
Aspergillus niger	10 mm	8 mm	15 mm
C. tropicalis	15 mm	14 mm	13 mm

Figure 4. 1-H NMR spectra of SHP

Figure 5. Zone of Inhibition against fungi

Figure 6. Zone of Inhibition against bacteria

Figure 1. F IR spectra of SHO

Figure 2. F IR spectra of SHP

Figure 3. 1-H NMR spectra of SHO

Figure 7. Comparative study

APPLICATION:

ANTIMICROBIAL ACTIVITY:

wo bacterial cultures (Fig. 6) including Proteus mirabilis (gram positive), Salmonella typi (gram negative) and two fungal cultures (Fig. 5) including Aspergillus niger and C. tropicalis were used to check the antimicrobial potential of both SHO and SHP. Amikacin was used as a control against bacterial cultures while Nystatin was used as a control against fungal cultures. he zone of inhibition against both bacteria and fungi were listed in the able 4. he bar graph shows the comparative study of SHO and SHP against certain microbes (Fig. 7).

CONCLUSION

We can infer from the results above that SHP was successfully prepared from the hexane extract of Salvia Hispanica and it was characterized by using F IR and ¹H NMR spectroscopy. he physico-chemical analysis of the SHP and phytochemical screening and basic radical identification of SHO were examined. he antimicrobial potential of the prepared polyol and the oil were tested against certain bacteria and fungi. hus, the prepared bio-polyol can be used as an alternative for organic polyols which can be effectively use for the preparation of certain bio- polymers.

CONFLICTS OF INTEREST he authors declare that they have no potential conflicts of interest.