Abstract
The purpose of this study was to investigate the chemical composition of the essential oil extracted from Psidium guajava leaves and to explore the medicinal value of β-caryophyllene in pharmaceutical science. The essential oils of P. guajava leaves were extracted by the hydrodistillation method, using a Clevenger-type apparatus and the chemical composition of essential oils was investigated by gas chromatography–mass spectrometry. The yield of extracted essential oils was 0.6% (w/w) dry weight. The main compounds found in the leaves were β-caryophyllene (20.34%), globulol (8.20%), trans-nerolidol (7.72%), aromadendrene (4.34%), cis-α-bisabolene (3.82%), tetracosane (3.68%), octadecane (3.66%), Z,Z,Z-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (3.44%), β-bisabolene (3.41%), limonene (3.09%), octacosane (2.88%), δ-cadinene (2.52%), and 1,4-cadadiene (2.04%). The main chemical class of the essential oil was terpenoids, which represent 71.65%, followed by hydrocarbons (26.31%). A total of 50 components were identified, among these β-caryophyllene was found to be dominant, which has great medicinal value, and some other compounds were also identified for the first time in the essential oil of P. guajava leaves.
Psidium guajava L. (family: Myrtaceae) is commonly known as guava. It is one of the most effective plants used for their therapeutic effects. 1 The leaves of this plant are used in traditional medicine for treatment of various ailments such as gastroenteritis, wounds, dysentery, lung problems, diarrhea rheumatism, and ulcer, whereas the leaf extracts have also been reported to show biological activities, including antitussive, antiallergic, antinociceptive, antioxidant, antidiarrheal, anti-inflammatory, hepatoprotective, antimicrobial, antiplasmodial, antispasmodic, and antidiabetic activities. 1 Some of the active ingredients such as guajaverin, guayfotavolic acid, guajadial, guavanoic acid, and other flavonoids are identified from this plant. 2–6
A variety of compounds are encountered in the essential oil (EO) of the leaves, including α-pinene, β-caryophyllene, limonene, veridiflorol, and nerolidol, and these EOs show antimicrobial, antioxidant, and antiproliferative activities. 1,7–11 There have been several reports on the leaf oil compositions of P. guajava from various locations around the world, and there is wide variation in the compositions of the EOs. 1
Essential oils are effective and active components of plants that can be extracted from different parts of plants. Plants’ EOs gained momentum because of contact insecticidal and fumigant activities. Essential oil is a potential candidate for weed control, and is being tried for pest and disease management. In addition, EOs are easily extractable, are eco-friendly as they are biodegradable and easily catabolized in the environment, do not persevere in water and soil, have little or no toxicity against vertebrates, mammals, birds, and fishes, and can be used in sensitive areas such as hospitals, restaurants, homes, and schools. 12,13
Currently, approximately 3 000 EOs, 300 of which are commercially important, are used extensively, especially in the pharmaceutical, agronomy, cosmetic, food, and perfume industries. In addition to these important properties, their chemical components attribute to a variety of effective biological activities, such as anticancer, antioxidant, anti-inflammatory, and antimicrobial. Essential oils are currently attracting interest in the scientific community and there is much research being conducted on their pharmacological activities; they are necessary in prevention and cure of microbial diseases and oxidative stress, including inflammations, viral infections, thrombosis, cardiovascular diseases, bacterial infections, and atherosclerosis. 14–18 In this work, we present gas chromatography–mass spectrometry (GC–MS) investigation of the EO components from P. guajava leaves obtained from Mirpurkhas, Pakistan, and compare the composition with several previous analyses.
The EO of P. guajava was yellowish with a special aroma. The yield of the EO extracted by the hydrodistillation method was 0.6% (w/w) on dry weight basis. With respect to previous reports, 1,7–11 yields are comparable, but little difference may be related with seasonal variation or growing habitats. 19,20
The results obtained from the GC–MS analysis are presented in Table 1, on the basis of their elution order on the HP-5 column. A total of 50 components were identified. The major constituents of the oils in our study were found to be β-caryophyllene (20.34%), globulol (8.20%), nerolidol 2 (7.72%), aromadendrene (4.34%), cis-α-bisabolene (3.82%), tetracosane (3.68%), octadecane (3.66%), Z,Z,Z-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (3.44%), β-bisabolene (3.41%), limonene (3.09%), octacosane (2.88%), δ-cadinene (2.52%), and 1,4-cadadiene (2.04%).
Gas Chromatography–Mass Spectrometry Investigation of Essential Oil Components From Psidium guajava Leaves.
RRI, relative retention indices; RRT, relative retention time; SD, standard deviation.
Considering the main chemical classes, the major class of the oil was terpenoids, which represent 71.65% of all identified compounds presented in Table 2. The content of monoterpene hydrocarbon was about 4.23% and diterpene hydrocarbon 2.85%. Oxygenated monoterpene content was 2.78%, including 2 compounds linalool and carvacrol. The content of sesquiterpene (oxygenated sesquiterpene and sesquiterpene hydrocarbon) was found to be 61.79% and oxygenated sesquiterpenes 7.78%. There was a huge amount of sesquiterpene hydrocarbons, with especially β-caryophyllene, globulol, and nerolidol 2 representing 20.34%, 8.20%, and 7.72%, respectively. Along with terpenoids, other hydrocarbons were also identified in the EOs. The total content of hydrocarbons was found to be 26.31% and the major hydrocarbon component was tetracosane (3.68%).
Composition of Grouped Components of Essential Oil of Psidium guajava Leaves.
NC, number of compounds.
Similarly, already reported studies of guava leaf EO indicated that the terpenoid class was found to be in a major concentration. The results of the present study were compared with the reported studies. 8,9,11,21
Comparative study of EO components present in the P. guajava leaves showed great variation in the concentration of β-caryophyllene. In the present study, the concentration of β-caryophyllene was found to be 20.34%, while that of β-caryophyllene (21.6%) and enerolidol (19.2%) was found higher as reported by Pino et al. 21 Similarly, limonene (42.1%) and β-caryophyllene (21.3%) were reported in higher concentrations by Ogunwande et al. 8 According to da Silva et al, 9 α-pinene (23.9%) and 1,8-cineole (21.4%) were found to be in greater concentrations. Khadhri et al 11 quoted that veridiflorol (36.4%) and trans-caryophyllene (5.9%) were dominant. Khadhri et al 11 reported the highest concentration of β-caryophyllene (27.7%) followed by α-pinene (14.7%). The quantitative variations among these results of different studies may be attributed to genetic variability and/or different geographic/environmental conditions. Different procedures used for the extraction of volatile components and different analytical procedures are also responsible for the variation among these results.
Some new components, such as tetracosane (3.68%), octadecane (3.66%), Z,Z,Z-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (3.44%), octacosane (2.88%), phytane (2.15%), nonacosane (2.03%), 2-methyl octadecane (1.57%), heptadecane (1.56%), caryophylladienol II (1.30%), 3-methyl octadecane (1.13%), 1-nonadecene (0.93%), 2-methyl eicosane (0.86%), eicosane (0.83%), 1-hexacosene (0.80%), tricosane (0.75%), p-allylanisole (0.71%), 4-methyl octadecane (0.71%), pristine (0.70%), 2-methyl heptadecane (0.57%), 1-tricosene (0.57%), γ-selinene (0.52%), heneicosane (0.35%), α-gurjunene (0.27%), β-guaiene (0.23%), γ-maaliene (0.17%), isoledene (0.13%), and β-gurjunene (0.11%), were identified. The names of all components are reported as such as per identification by National Institute of Standards and Technology Mass Spectral (NIST MS) library installed in the GC–MS. Although it is very difficult to separate enantiomers by GC–MS without using a chiral column, due to better performance of GC column and temperature programming, some enantiomers present in the EO of P. guajava leaves belonging to Riyali variety are separated and identified by MS library.
To our best knowledge this is the first time that 50 components reported in the EOs of P. guajava leaves and a detailed composition of the EOs of P. guajava leaves are studied in Pakistan.
β-Caryophyllene is the main constituent of the EOs in this study. It has been found that β-caryophyllene has important medicinal value. For instance, it is widespread in plants, is a food additive approved by the Food and Drug Administration, and ingested daily with food. This compound is capable of changing the inflammatory processes in humans through the endocannabinoid system. β-Caryophyllene does not bind to the central cannabinoid receptor type 1, and, therefore, does not exert psychoactive effects. 22
β-Caryophyllene is generally dispersed in the EOs of various plants (Myrica gale, Eugenia caryophyllata, and Comptonia peregrina). Numerous biological activities, such as local anesthetic, anticarcinogenic, antioxidant, antibiotic, and anti-inflammatory, 23 anxiolytic and antialcoholic, 24 antidepressant, 25 neuroprotective, 26 and antinociceptive activities, 27 are recognized for β-caryophyllene.
According to a work reported on β-caryophyllene, this compound could increase the intracellular accumulation of anticancer agents, thereby potentiating their cytotoxicity due to the absorption of 5-fluorouracil across human skin. β-Caryophyllene facilitates the passage of paclitaxel through membranes and thus potentiates its anticancer activity. 23
The GC–MS analysis of the hydrodistilled extracted EO (0.6% (w/w)) of P. guajava leaves explored about 27 new components out of 50 with a high content of sesquiterpene hydrocarbon (48.76%). Among these the main component was β-caryophyllene. Due to the high content of β-caryophyllene, it can be concluded that the leaves of this plant have potential use in cancer treatment.
Experimental
Plant Material Collection
The leaves of P. guajava were obtained from mature trees at Mirpurkhas (longitude: 25.4725°N and latitude: 68.7376°E), Sindh Province, Pakistan in January 2016. Voucher specimen was deposited in the Herbarium of Institute of Plant Sciences, University of Sindh Jamshoro, under the code 53 141. The plant was identified as a Riyali variety by the taxonomist of the same institution.
Essential Oil Isolation
The air-dried and ground leaves were subjected to hydrodistillation for 3 hours using a Clevenger-type apparatus according to European Pharmacopoeia, 1996. The obtained EO was concentrated on a rotary evaporator and then the concentrated sample was dried over anhydrous sodium sulfate for 1.5 hours. The sample was filtered and the remaining n-hexane was evaporated under nitrogen flow, and stored in sealed vials at +4°C until analyzed by GC–MS.
GC–MS Chemical Analysis
The EOs were analyzed using an Agilent 6890 n gas chromatograph equipped with a HP-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm) coupled with MS-5975. Essential oil sample was diluted in n-hexane and 1 µL volume injected by autosampler injector at a carrier gas helium flow of 1 mL/min with a split ratio of 1:50. Temperature programming: (a) initial oven temperature 80°C, holding time 5 min; (b) increased to 130°C at the rate of 8° C/min; (c) then rose to 200°C at the rate of 5 °C/min; (d) finally rose to 280°C at the rate of 15°C/min, holding time 4 min. Ion source temperature was 230°C and electron impact ionization was 70 eV.
Identification of EO
Individual compounds of the EOs of the guava leaves were identified by using their mass spectra, and relative retention indices relative to n-alkanes (C8-C29). These components were also identified by comparing the obtained mass data with NIST MS library, and the matching percentage selected was above 90%. The relative percentage was calculated from peak area of the gas chromatogram.
Statistical Analysis
Each sample was injected 3 times of repeated extraction of EOs by hydrodistillation. The average of the results obtained thrice was expressed as mean ± standard deviation.
Footnotes
Acknowledgments
The authors are grateful for the financial support provided by the National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Financial support was provided by the National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro.