Spectroscopic and chromatographic evaluation of solvent extracted guava seed oil

ABSTRACT

The aim of present study was to extract the guava seed oil for its quantitative determination and to evaluate chemical characteristics including fatty acid composition and to compare with reported literature. Average oil content of guava seeds was found to be 11.12 g oil /100g seed. The iodine value, acid value, free fatty acid, peroxide value and saponification value were found to be 120.55 g of I₂/100 g oil, 3.74 g/100g of oil, 1.86 g/100 g oil, 4.13 mEq/kg of oil and 190.74 mg/100 g of oil, respectively. Fatty acid composition was evaluated by gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy. By GC-MS, a total of eighteen fatty acids were identified from which twelve were saturated fatty acids (SFAs) and six were unsaturated fatty acids (USFAs). Among SFAs, palmitic acid, stearic acid and arachidic acid were dominant, while linoleic and oleic acids were main USFAs found in guava seed oil. GC-MS results indicated that guava seed oil is a good source of essential fatty acids.

Abbreviations: FA: Fatty acid; FAMEs: Fatty acid methyl ester; PUFA: Polyunsaturated fatty acids; SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids;

KEYWORDS:

• Chemical characteristics
• Fatty acid profile
• FT-IR
• GC-MS
• Oil extraction
• Psidium guajava

Introduction

Psidium guajava L. (genus: Psidium; family: Myrtaceae) is a perennial tree that grows in tropical and subtropical areas of the world.^[1,2] Psidium guajava is growing in India, Pakistan, Brazil, Mexico, Bangladesh, USA, Thailand and number of other countries. The main world producers of guava are India and Mexico. Pakistan is the fourth major country in production of guava fruit (0.55 million tonnes per year).^[3] In Pakistan, guava (locally called Amrood) is grown widely in the province of Sindh and Punjab. After citrus and mango fruits, the guava has third position with respect to cultivation and area. In Sindh province, Larkana is the largest guava producing district followed by Mirpurkhas, Hyderabad, Nawabshah and Nausheroferoz. Guava fruit is extremely nutritious, delicious in taste, low price and fruit produced round the year.^[4]

Psidium guajava is a well-known medicinal plant which contains number of phytochemicals. Its different parts are used in various indigenous systems of medicine and health such as cholera, bowels, ulcers and wounds.^[5] Leaves extract of guava tree are used to cure number of disease such as anti-microbial, anti-inflammatory, anti-oxidant and anti-cough effects.^[6] A decoction of leaves is used for the treatment of cholera, vomiting and diarrhoea.^[7] Leaves contain tannin, resin, eugenol, essential oil, caryophyllene, terpenoids and flavonoids.^[8] The plant bark is used as an astringent as well as antidiarrhoeatic in children.^[9]

Guava fruit is high in vitamins and minerals such as vitamin E, vitamin C (Ascorbic acid), vitamin A, niacin (vitamin B3), thiamine (vitamin B3) and Fe, Ca, K, P, Mg, etc. Guava fruit also has important medicinal properties such as anti-cancer, sedative, digestive, antispasmodic, carminative, antihyperglycemic, antipyretic, anti-bacterial, anti-inflammatory and analgesic activities.^[10,11] Various chemical constituents of guava fruit have been identified such as phenolic compounds (tannins and phenols), carotenoids, triterpenes, fatty acids, flavonoids, saponins, vitamins, essential oils and lectins.^[12]

Guava seed extract has been reported as corrosion inhibitor (decreases corrosion rate of a material) for carbon steel in HCl (acidic) medium.^[13] It has been reported that guava seed posses significant amount of tryptophan, methionine, linoleic acid and fiber content confines its use as wheat flour alternate in the preparation of cookies.^[14] The plants contain a range of fatty acids with different structure commonly found in edible oils, and some of them are highly beneficial for health.^[15]

The skin and seeds of guava fruit contain carotenoids, glycosides, flavonoid and phenolic compounds. They are also rich in other nutrients, essential fatty acids, and contain good amount of iodine. The fruits are very rich in seeds, which contain 14 % oil content (on dry weight), with 13% starch and 15% proteins.^[15] Guava oil has moisturizing retention properties due to the presence of lycopene (powerful antioxidant).^[16]

Guava oil contains fatty acid such as palmitic acid, stearic acid, oleic acid, linoleic acid and linoleic acid .^[15–18] Omega-6 (linoleic acid) is main fatty acid identified in guava seed oil. Linoleic acid provides oleochemical uses in oil paints, surfactants, lubricants, varnishes, cosmetics and pharmaceuticals.^[19] Saturated fatty acids such as palmitic acid and stearic acid are used in manufacturing of soaps, cosmetics, lubricants and softeners.^[20] Therefore, oil present in guava seed oil further requires improvements in fatty acid compositions. The objective of present study was to assess the chemical characteristics including fatty acid composition of guava seed oil collected from Mirpurkhas, Sindh, Pakistan by chromatographic and spectroscopic techniques and to compare results with published literature.

Material and methods

Collection of plant material

The mature fruit of Psidium guajava was collected from Mirpurkhas, Sindh, Pakistan (longitude: 25.4725° N and latitude: 68.7376° E) in March 2015. The plant was deposited (voucher specimen, 53141) in the herbarium of Institute of Plant Sciences, University of Sindh, Jamshoro, Pakistan. The plant was identified by taxonomist of same institution. The seeds were separated from fruit, cleaned and dried in shadow. The seeds were ground (using grinder), and oil was extracted using organic solvent.

Extraction of guava seed oil

Extraction of oil from guava seeds was carried out by Soxhlet extraction method. Around 30 g powdered guava seeds were taken in a cellulose thimble and placed in a Soxhlet apparatus using n-hexane as an oil extracting solvent. The n-hexane extract was concentrated by a rotary evaporator under reduced pressure to obtain crude oil. The sample was kept under fume hood to evaporate remaining n-hexane from oil. The percentage of oil content was reported on dry basis of guava seeds.

Moisture content and chemical analysis

Moisture content of guava seed was determined by oven drying at 105°C for 3 h according to AOAC method.^[21] The results of moisture content of seeds expressed in percentage. The iodine value (IV), acid value (AV), free fatty acid value (FFA), peroxide value (PV) and saponification value (SV) were determined by Official Method (AOCS, 1992).^[22]

Preparation of fatty acid methyl ester (fames)

FAMEs of guava seed oil were prepared by adding 20 mL of methanol two pellets of potassium hydroxide along with 20 mg of guava seed oil in round bottom flask. The mixture was refluxed about half an hour on heating mental and then cooled reaction mixture at room temperature. The reaction mixture was transferred to the separatory funnel and added 10 mL of hexane and 20 mL of water and shaken vigorously to separate aqueous and organic layer. The organic layer (hexane) was separated and dried over anhydrous sodium sulphate then analysed by GC-MS according to reported procedure.

Fourier transform infrared (FT-IR) spectroscopic analysis

FT-IR analysis of FAMEs of guava seed oil was performed using Thermo Nicolet 5700 spectrometer (Thermo Nicolet Analytical Instruments, Madison, WI) equipped with removable ZnSeattenuated total reflectance (ATR) accessory and deuterated triglycine sulfate (DTGS) detector. The FT-IR spectrum of guava seed oil FAMEs was recorded (at room temperature, 25°C) in the mid infrared range 4000–650 cm⁻¹ at 32 scans with resolution of 4 cm⁻¹. Spectrum of sample was obtained against background of air spectrum. After recording spectrum, ATR crystal (sample compartment) was carefully cleaned with organic solvents (hexane and methanol).

Analysis of fatty acids by GC-MS

Finally, FAMEs of guava seed oil were analysed on GC-MS instrument (Agilent 6890 N GC) attached with mass selective detector (MS-5975 inert XL), injector (auto sampler 7683-B) and column (HP-5MS; 30 m × 0.25 mm i.d. film thickness 0.25 µm). Helium was used as a carrier gas with flow rate of 1.5 mL/min. About 1.0 µL of sample was injected, using split mode (split ratio, 1:50). The MS was operated in the electron impact mode (EI) at 70 eV using 50–550 m/z scan range. Initial oven temperature was set at 90°C for 5 min, raised to 220°C at 5°C/min (2 min hold) and then increased to 270°C at 5°C/min (5 min hold). The injector and MS transfer line temperature were set at 220–290°C, respectively.

Identification of fatty acids and statistical analysis

Individual fatty acid of guava seed oil was identified by matching their mass spectra of known compounds (> 90%) in database incorporated in GC-MS software provided by NIST (National Institute of Standards and Technology) and Wiley. The percent area (relative %) of fatty acids was calculated from the peak areas. FAMEs of guava seed oil were analysed in triplicate and results expressed as mean ± standard deviation (SD).

Results and discussion

The moisture content of guava seed in present study was found to be 8.33g/100 g. Moisture content is important parameter in determining the shelf life and storage condition of seeds. Guava oil was extracted from seeds by Soxhlet extraction method using n-hexane as extraction solvent. Total oil content of Psidium Guajava seeds was found to be 11.12 g of oil /100 g of seed on dry bases. The oil content found in present study is higher than Habib^[23] 9.1 g of oil /100 g of seed and Opute^[24] 9.4 g of oil/100 g of seed, whereas slightly lower than Uchôa-thomaz et al.^[12] 13.93 g of oil/100 g of seed^[12]. To check the quality of guava seed oil, iodine value (IV), acid value (AV), free fatty acids value (FFA), peroxide values (PV) and saponification value (SV) were calculated as shown in Table 1. The IV is a measurement of degree of unsaturation and reflects the susceptibility of oil to oxidation. In present study, oil contains high IV (120.55 g I₂/100g oil), indicating that oil is composed of mainly unsaturated fatty acids. Acid value was found to be (3.74 g/100 g oil) indicating the content of FFA in oil. It is an important variable when considering the quality of oil. FFA value was found to be (1.86 g/100 g oil). Generally, it is considered that lower the value of FFA better the oil quality. PV is the measure of rancidity index of oil, and it was observed that guava seed oil contains (4.13mEq/kg of oil) PV. Higher level of PV is the indication of a rancid oil/fat, but in guava seed oil, PV is relatively low which indicates that there is less chance of rancidity. The SV of guava seed oil was found to be (190.74 mg/100 g oil) which indicated that oil contains high-molecular weight fatty acids. The obtained SV of guava seed oil is almost comparable with most of the vegetable oils. Fatty acid composition and characterization of guava seed oil were evaluated by chromatographic and spectroscopic methods. To analyse fatty acid composition of guava seed oil, the oil was subjected to preparation of FAMEs. The esters were analysed by different techniques such as FT-IR and GC-MS.

Table 1. Chemical parameters of guava seed oil.

Chemical Parameters	Guava seed oil
Iodine value (g of I2/100g of oil)	120.55 ± 0.05
Acid value (g/100g of oil)	3.74 ± 0.07
Free fatty acid value (g/100g of oil)	1.86 ± 0.03
Peroxide value (4.13mEq/Kg of oil)	4.13 ± 0.02
Saponification value (mg of KOH/100g of oil)	190.74 ± 0.04

FT-IR spectral analysis of FAMEs of guava seed oil confirmed the presence of C = O and C-O characteristic bands at 1742 cm⁻¹ and 1169 cm⁻¹. Bands due to unsaturated fatty acids at 3009 cm⁻¹ and 1664 cm⁻¹were also observed as shown in Fig. 1. FT-IR spectral data supported the results FAMEs obtained by GC-MS. The GC-MS results of guava seed oil provided number of fatty acids that turned out to be different from previously published data.^[12] The results revealed that a total of 18 fatty acids methyl esters were separated and quantified based on their elution order as shown in Fig. 2.

Figure 1. FTIR spectrum of guava seed oil FAMEs.

Figure 2. GC-MS chromatogram for fatty acids obtained from guava seed oil.

Among 18 fatty acids, twelve saturated (26.45%) and six unsaturated (73.55%) fatty acids were identified as shown in Table 2. Among unsaturated fatty acids, the predominant fatty acids were linoleic acid (60.03%) and oleic acid (12.57%), whereas palmitic acid (14.81%), stearic acid (9.08%) and arachidic acid (1.31%) found as a main saturated fatty acids.

Table 2. Oil content (%) and fatty acid composition (%) of P. guajava seed oil by GC-MS.

		Present work	Reported literature
Fatty acids	Carbon number	Area, % ±SD	^[Citation12]	^[Citation24]	^[Citation23]
Myristic acid	C14:0	0.21 ± 0.02	0.10	-	0.89
Pentadecanoic acid	C15:0	0.03 ± 0.05	-	-	1.20
Palmitoleic acid	C16:1	0.13 ± 0.02	-	-	-
Palmitic Acid	C16:0	14.81 ± 0.02	8.00	9.7	13.3
Margaric acid	C17:0	0.19 ± 0.04	0.07	-	-
Oleic acid	C18:1	12.57 ± 0.09	9.42	7.8	14.0
Stearic acid	C18:0	9.08 ± 0.06	4.48	3.4	11.1
Linoleic acid	C18:2	60.03 ± 0.04	77.35	79.1	52.1
Nonadecanoic acid	C19:0	0.04 ± 0.02	-	-	-
Methyl ricinoleate	C18:1	0.21 ± 0.03	-	-	-
10,13-Eicosadienoic acid	C21:2	0.12 ± 0.01	-	-	-
11-Eicosenoic acid	C21:1	0.49 ± 0.01	-	-	-
Arachidic acid	C20:0	1.31 ± 0.08	0.12	-	-
Heneicosanoic acid	C21:0	0.07 ± 0.01	0.14	-	-
Behenic acid	C22:0	0.33 ± 0.02	0.10	-	-
Tricosanoic acid	C23:0	0.08 ± 0.01	-	-	-
Lignoceric acid	C24:0	0.23 ± 0.01	-	-	-
Cerotic acid	C26:0	0.07 ± 0.06	-	-	-
ΣSFA		26.45	13.01	13.1	26.49
ΣMUFA		13.40	9.42	7.8	14.0
ΣPUFA		60.15	77.35	79.1	52.1
ΣUSFA		73.55	86.77	86.9	66.1
Oil content (g/100g)		11.12 ± 0.03	13.93	9.4	9.1

SD, standard deviation, SFA, saturated fatty acids; MUFS, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; UFA, unsaturated fatty acids.

Previous study, on guava seed oil, revealed the identification of lauric acid, myristic acid, palmitic acid, heptadecanoic acid, stearic acid, oleic acid (n-9), linoleic acid (n-6), arachidic acid, gondoic acid, linoleic acid and behenic acid.^[12] Habib^[23] reported that high levels of unsaturated fatty acids (86.9%) mainly linoleic acid (79.1%) and oleic acid (7.8%) were identified by Opute.^[24] In present study, on guava seed oil showed some new fatty acids which has been identified first time in guava seed oil such as palmitoleic acid, nonadecanoic acid, methyl ricinoleate, 10,13-eicosadienoic acid, 11-eicosenoic acid, heneicosanoic acid, tricosanoic acid, lignoceric acid and cerotic acid in minor concentration. The major unsaturated fatty acids such as linoleic acid and oleic acid were found to be common, but in different concentration from previously reported studies.^{[11,12,23,24]} The possible reason between obtained results and reported data may be due to either geographical location or variety or the extraction technique employed.^[24]

Comparative results of fatty acids classes in Table 2 indicated that highest concentration of SFAs was reported by Habib^[23] (26.49%). In comparison with other reported works, our finding of SFA (26.45%) is higher than Opute^[24](13.1%) and Uchôa-thomaz et al.,^[12](13.01%). On the other hand, higher concentrations of USFAs were observed by Uchôa-thomaz et al.,^[12] (86.77%) and Opute^[24] (86.9%) but in present study (73.55%) that is higher than Habib^[23] (66.1%). While these results are comparatively lower than the findings of Opute^[24] (86.9%) and Uchôa-thomaz et al .,^[12] comparative results of the major fatty acids are shown in Table 2 which indicates that highest concentration of linoleic acid was reported by Opute^[24] (79.1 %), followed by Uchôa-thomaz et al^[12](77.35%), present study (60.03%) and Habib^[23] (52.10%). Whereas other major fatty acid was oleic acid, maximum concentration of oleic acid reported by Habib^[23] (14.0%), followed by present study (12.57%), Uchôa-thomaz et al^[12] (9.42%) and Opute^[24](7.80%). The highest concentration of SFA includes stearic acid, which was reported by Habib^[23](11.10%), followed by present study (9.08%), Uchôa-thomaz et al^[12](4.48%) and Opute^[24](3.40%). On the other hand, palmitic acid was observed as a major saturated fatty acid by present study (14.81%), followed by Habib^[23] (13.30%), Opute^[24](9.70%) and Uchôa-thomaz et al^[12] (8.00%).

General classification of guava seed oil with regard to fatty acids discussed in Table 2 has been found in the sequence of MUFA (13.40%) < SFA (26.45%) < PUFA (60.15%). The minimum ratio of PUFA/SFA set by the department of Health UK is 0.45. In this study, the ratio of PUFA/SFA found 2.27, which was much higher than the recommended value. In comparison with the published data on guava seed oil, this ratio is much lower than the reported value of 8.11.^[12] This variation is due to the presence of higher concentrations of PUFA.

From the identified components of guava seed, linoleic acid and oleic acid are particular importance because of their many properties and uses. The presence of these fatty acids in high quantity makes guava seed oil beneficial to human health, especially for heart patients. Therefore, guava seed oil can be considered as a good source of linoleic acids and has a wide range of applications in the nutrition, cosmetics and medicine. On the other hand, the results of this study are also useful for phytopharmaceutical industries to establish their quality control profile. This study helps to understand that after extracting the juice seeds could be utilized in above mentioned applications.

Conclusion

Based on our findings, it is recommended that extraction of oil from guava seeds should be carried out on industrial scale for food, cosmetic and pharmaceutical industries. The fatty acid composition of guava seed oil varies from different geographic locations as reported in the literature, but in all studies, linoleic acids has been reported as dominant fatty acid, and it has number of application. More harvesting of Psidium guajava may increase production of health beneficial fruit and better environment as well as greater production of valuable oil from the seeds.

Declaration of interest

The authors have declared no conflict of interest.

Funding

The authors are grateful for the financial support provided by National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro.

Additional information

Funding

The authors are grateful for the financial support provided by National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro.