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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.

Keywords

leaves essential oils GC–MS β-caryophyllene

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%).

Table 1

Gas Chromatography–Mass Spectrometry Investigation of Essential Oil Components From Psidium guajava Leaves.

RRT	RRI	Name	Relative, % ±SD
3.50	937	α-Pinene	0.33 ± 0.06
4.29	981	β-Myrcene	0.05 ± 0.02
5.06	1032	Limonene	3.09 ± 0.05
5.13	1046	Eucalyptol	2.24 ± 0.07
5.37	1051	β-cis-Ocimene	0.05 ± 0.03
6.48	1101	Linalool	0.30 ± 0.09
8.38	1177	Terpinen-4-ol	0.07 ± 0.01
8.87	1199	p-Allylanisole	0.71 ± 0.03
11.18	1298	Carvacrol	0.17 ± 0.02
13.38	1353	α-Cubebene	1.93 ± 0.01
14.23	1408	α-Gurjunene	0.27 ± 0.01
14.59	1417	β-caryophyllene	20.34 ± 0.04
14.70	1428	γ-Maaliene	0.17 ± 0.08
14.80	1439	β-Gurjunene	0.11 ± 0.07
14.99	1440	Aromadendrene	4.34 ± 0.02
15.33	1456	Z,Z,Z-1,5,9,9-Tetramethyl-1,4,7-cycloundecatriene	3.44 ± 0.01
15.49	1457	Alloaromadendrene	1.35 ± 0.08
15.76	1470	γ-Cadinene	0.21 ± 0.04
15.84	1477	γ-Muurolene	0.76 ± 0.01
16.10	1489	β-Selinene	0.42 ± 0.02
16.16	1490	β-Guaiene	0.23 ± 0.09
16.46	1504	cis-α-Bisabolene	3.82 ± 0.03
16.62	1506	β-Bisabolene	3.41 ± 0.02
16.98	1519	δ-Cadinene	2.52 ± 0.08
17.03	1528	Isoledene	0.16 ± 0.06
17.95	1564	Nerolidol 2	7.72 ± 0.03
18.03	1570	γ-Selinene	0.52 ± 0.04
18.48	1580	Globulol	8.20 ± 0.02
18.88	1588	Ledol	1.88 ± 0.08
19.63	1637	Caryophylladienol II	1.30 ± 0.02
20.01	1679	α-Cadinol	1.36 ± 0.06
20.65	1682	α-Bisabolol	0.77 ± 0.05
20.87	1700	Heptadecane	1.56 ± 0.06
20.99	1707	Pristane	0.70 ± 0.03
22.21	1764	2-Methyl heptadecane	0.57 ± 0.09
23.02	1800	Octadecane	3.66 ± 0.04
23.20	1811	Phytane	2.15 ± 0.02
24.17	1860	4-Methyl octadecane	0.71 ± 0.06
24.28	1887	2-Methyl octadecane	1.57 ± 0.02
24.44	1890	3-Methyl octadecane	1.13 ± 0.03
27.20	1899	1-Nonadecene	0.93 ± 0.03
28.52	1990	2-Methyl eicosane	0.86 ± 0.06
29.81	2000	Eicosane	0.83 ± 0.02
34.64	2100	Heneicosane	0.35 ± 0.05
34.77	2294	1-Tricosene	0.57 ± 0.02
35.50	2300	Tricosane	0.75 ± 0.03
37.04	2400	Tetracosane	3.68 ± 0.02
37.50	2596	1-Hexacosene	0.80 ± 0.04
37.90	2800	Octacosane	2.88 ± 0.09
41.19	2900	Nonacosane	2.02 ± 0.03

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%).

Table 2

Composition of Grouped Components of Essential Oil of Psidium guajava Leaves.

Class	Name	NC	%
Terpenoids	Monoterpene hydrocarbon	05	4.23
	Diterpene hydrocarbon	02	2.85
	Oxygenated monoterpene	04	2.78
	Sesquiterpene hydrocarbon	17	48.76
	Oxygenated sesquiterpene	05	13.03
Hydrocarbon		17	26.31
Total %		50	97.96

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 al¹¹ quoted that veridiflorol (36.4%) and trans-caryophyllene (5.9%) were dominant. Khadhri et al¹¹ 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.

References

Satyal

Paudel

Lamichhane

Setzer

. Leaf essential oil composition and bioactivity of Psidium guajava from Kathmandu, Nepal. Am J Essent Oil Nat Prod. 2016;3(2):11–14.

Jiménez-Escrig

Rincón

Pulido

Saura-Calixto

. Guava fruit (Psidium guajava L.) as a new source of antioxidant dietary fiber. J Agric Food Chem. 2011;49(11):5489–5493.doi:10.1021/jf010147p

Begum

Hassan

SI.

Ali

SN.

Siddiqui

. Chemical constituents from the leaves of Psidium guajava . Nat Prod Res. 2004;18(2):135–140.doi:10.1080/14786410310001608019

Soares

FD.

Pereira

Marques

MO.

Monteiro

. Volatile and non-volatile chemical composition of the white guava fruit (Psidium guajava) at different stages of maturity. Food Chem. 2007;100(1):15–21.doi:10.1016/j.foodchem.2005.07.061

Gutiérrez

RM.

Mitchell

Solis

. Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. J Ethnopharmacol. 2008;117(1):1–27.doi:10.1016/j.jep.2008.01.025

Wang

Chen

YH.

Zhang

Deng

Zou

. Chemical components and bioactivities of Psidium guajava . Int J Food Sci Nutr. 2014;5:98–114.

Garraffo

HM.

Pannell

. The essential oil of the leaves of Psidium guajava L. J Essent Oil Res. 1991;3(3):187–189.

Ogunwande

IA.

Olawore

NO.

Adeleke

KA.

Ekundayo

Koenig

. Chemical composition of the leaf volatile oil of Psidium guajava L. growing in Nigeria. Flavour Fragr J. 2003;18(2):136–138.doi:10.1002/ffj.1175

da Silva

JD.

Luz

AI.

da Silva

et al . Essential oils of the leaves and stems of four Psidium spp. Flavour Fragr J. 2003;18(3):240–243.doi:10.1002/ffj.1219

10.

Adam

Vahirua-Lechat

Deslandes

Menut

. Aromatic plants of French Polynesia. V. Chemical composition of essential oils of leaves of Psidium guajava L. and Psidium cattleyanum Sabine. J Essent Oil Res. 2011;23(1):98–101.

11.

Khadhri

El Mokni

Almeida

Nogueira

JM.

Araújo

. Chemical composition of essential oil of Psidium guajava L. growing in Tunisia. Ind Crops Prod. 2014;52:29–31.doi:10.1016/j.indcrop.2013.10.018

12.

Raju

KC.

Chalannavar

RK.

Narayanaswamy

VK.

Baijnath

Odhav

. Chemical composition of essential oil of Psidium cattleianum var. lucidum (Myrtaceae). Afr J Biotechnol. 2012;11(33):8341–8347.doi:10.5897/AJB10.1942

13.

Ud-Daula

AFMS.

Demirci

Abu Salim

et al . Chemical composition, antioxidant and antimicrobial activities of essential oils from leaves, aerial stems, basal stems, and rhizomes of Etlingera fimbriobracteata (K.Schum.) R.M.Sm. Ind Crops Prod. 2016;84:189–198.doi:10.1016/j.indcrop.2015.12.034

14.

Sepahvand

Delfan

Ghanbarzadeh

Rashidipour

Veiskarami

GH.

Ghasemian-Yadegari

. Chemical composition, antioxidant activity and antibacterial effect of essential oil of the aerial parts of Salvia sclareoides . Asian Pac J Trop Med. 2014;7(Suppl 1):S491–S496.doi:10.1016/S1995-7645(14)60280-7

15.

Yagi

Babiker

Tzanova

Schohn

. Chemical composition, antiproliferative, antioxidant and antibacterial activities of essential oils from aromatic plants growing in Sudan. Asian Pac J Trop Med. 2016;9(8):763–770.doi:10.1016/j.apjtm.2016.06.009

16.

Mothana

RA.

Al-Said

MS.

Al-Yahya

MA.

Al-Rehaily

AJ.

Khaled

. GC and GC/MS analysis of essential oil composition of the endemic Soqotraen leucas virgata Balf.f. and its antimicrobial and antioxidant activities. Int J Mol Sci. 2013;14(11):23129–23139.doi:10.3390/ijms141123129

17.

Chen

Zhou

Deng

Zeng

. Chemical compositions and antibacterial activities of essential oils extracted from Alpinia guilinensis against selected foodborne pathogens. Ind Crops Prod. 2016;83:607–613.doi:10.1016/j.indcrop.2015.12.063

18.

Shakeri

Khakdan

Soheili

Sahebkar

Shaddel

Asili

. Volatile composition, antimicrobial, cytotoxic and antioxidant evaluation of the essential oil from Nepeta sintenisii Bornm. Ind Crops Prod. 2016;84:224–229.doi:10.1016/j.indcrop.2015.12.030

19.

Matias

EF.

Alves

EF.

Silva

et al . Seasonal variation, chemical composition and biological activity of the essential oil of Cordia verbenacea DC (Boraginaceae) and the sabinene. Ind Crops Prod. 2016;87:45–53.doi:10.1016/j.indcrop.2016.04.028

20.

Dhouioui

Boulila

Chaabane

Zina

MS.

Casabianca

. Seasonal changes in essential oil composition of Aristolochia longa L. ssp. paucinervis Batt. (Aristolochiaceae) roots and its antimicrobial activity. Ind Crops Prod. 2016;83:301–306.doi:10.1016/j.indcrop.2016.01.025

21.

Pino

JA.

Ortega

Rosado

. Volatile constituents of guava (Psidium guajava L.) fruits from Cuba. J Essen Oil Res. 1999;11(5):623–628.

22.

Corey

EJ.

Mitra

RB.

Uda

. Total synthesis of d,l-caryophyllene and d,l-isocaryophyllene. J Am Chem Soc. 1964;86(3):485–492.doi:10.1021/ja01057a040

23.

Legault

Pichette

. Potentiating effect of β‐caryophyllene on anticancer activity of α‐humulene, isocaryophyllene and paclitaxel. J Pharm Pharmacol. 2007;59(12):1643–1647.doi:10.1211/jpp.59.12.0005

24.

Katsuyama

Mizoguchi

Kuwahata

et al . Involvement of peripheral cannabinoid and opioid receptors in β‐caryophyllene‐induced antinociception. Eur J Pain. 2013;17(5):664–675.doi:10.1002/j.1532-2149.2012.00242.x

25.

Guimarães-Santos

Santos

DS.

Santos

et al . Copaiba oil-resin treatment is neuroprotective and reduces neutrophil recruitment and microglia activation after motor cortex excitotoxic injury. Evid Based Complement Alternat Med. 2012;2012(10):1–9.doi:10.1155/2012/918174

26.

Bahi

Al Mansouri

Al Memari

Al Ameri

Nurulain

SM.

Ojha

. β-Caryophyllene, a CB2 receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice. Physiol Behav. 2014;135:119–124.doi:10.1016/j.physbeh.2014.06.003

27.