SUPPLEMENTARY MATERIAL

Chemical composition and antimicrobial activity of the essential oil of senescent leaves of Guava
(Psidium guajava L.)
Trishna Chaturvedi1, Swati Singh2, Indrajeet Nishad3, Ajay Kumar4, Neha Tiwari3, Sudeep Tandon2,
Dharmendra Saikia4, Ram Swaroop Verma2*
1
Department of Genetics and Plant Breeding, CSIR-Central Institute of Medicinal and Aromatic Plants,
P.O. CIMAP, Lucknow, India
2
Department of Process Chemistry and Technology, CSIR-Central Institute of Medicinal and Aromatic
Plants, P.O. CIMAP, Lucknow, India
3
Department of Plant Pathology, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP,
Lucknow, India
4
Department of Molecular Bioprospection, CSIR-Central Institute of Medicinal and Aromatic Plants,
P.O. CIMAP, Lucknow, India
*
Corresponding author: e-mail: rs.verma@cimap.res.in

Abstract: The aim of the study was to investigate the chemical composition and antimicrobial activity of
the essential oil of the senescent leaves of Psidium guajava L. (Myrtaceae). The hydrodistilled essential
oil of the leaves was analysed by GC-FID and GC-MS. The antimicrobial activity of the oil was assessed
against human and plant pathogenic microorganisms by disc diffusion, microdilution broth and poison
food techniques. A total of forty-six constituents, representing 90.9% of the total oil composition were
identified. Major constituents of the oil were limonene (29.1%), (E)-caryophyllene (15.7%),
caryophyllene oxide (8.8%), caryophylla-4(12),8(13)-dien-5-ol (6.5%), (E)-nerolidol (4.0%), α-cadinol
(3.4%), and muurola-4,10(14)-dien-1-β-ol (2.5%). The chemical composition of the examined essential
oil was quite different from those reported earlier. The essential oil showed significant inhibition of
human pathogenic bacteria (MIC: 0.065–0.261 mg/mL) and plant pathogenic fungi namely Curvularia
lunata (86.02 %) and Fusarium chlamydosporum (82.80 %).

Keywords: Psidium guajava, senescent leaves, essential oil, limonene, human pathogen, plant pathogen

Experimental
Plant material and extraction of essential oil
The senescent leaves of Psidium guajava were collected from the experimental farm of CSIR-Central
Institute of Medicinal and Aromatic Plants, Lucknow during October, 2018. The plant material was
authenticated at Botany and Pharmacognosy Department, CSIR-Central Institute of Medicinal and

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Aromatic Plants, Lucknow (voucher specimen no: 8309). The leaves were shade dried for one week and
then hydrodistilled in a Clevenger-type apparatus for 3 h to extract the essential oil. The extracted
essential oil was dried over anhydrous sulphate and kept in cool place until further analysis.

Gas chromatography (GC) analysis

The essential oil analysis was carried out using GC-FID and GC-MS techniques. Briefly, quantification of
the essential oil constituents was done on Centurion Scientific Gas Chromatograph (model CS-5800),
equipped with flame ionization detector (FID) and BP-5 fused silica capillary column (5% phenyl)-
polymethylsiloxane stationary phase; 30 m  0.25 mm internal diameter; film thickness 0.25 µm). Oven
temperature was programmed from 60°C to 240°C with increase of 3°C min-1 and final hold time of 10
min. The injector and detector temperatures were 240°C and 250°C, respectively. Nitrogen was used as
carrier gas at 1.0 mL min-1. The injection volume was 0.6 µL (diluted in hexane: 1:6) with a split ratio of
1:60.

Gas chromatography-Mass spectrometry (GC-MS) analysis

GC-MS analysis of the oil sample was carried out on a Clarus 680 GC interfaced with a Clarus SQ 8C
Quadrupole mass spectrometer of PerkinElmer fitted with Elite-5 MS fused-silica capillary column (30 m
× 0.25 mm i.d., film thickness 0.25 µm). The oven temperature program was from 60 to 240°C, at 3°C
min-1, and programmed to 270°C at 5°C min-1. Injector temperature was 250 °C; transfer line and source
temperatures were 220 °C; injection size 0.03 µL neat; split ratio 1:50; carrier gas He at 1.0 mL min-1;
ionization energy 70 eV; mass scan range 40-450 amu.

Identification of constituents
Identification of the essential oil constituents was carried out on the basis of retention index (RI),
determined with reference to homologous series of n-alkanes (C7-C30), MS Library search (NIST and
WILEY), and by comparing RI and mass spectral data with the literature (Adams, 2007). The relative
amounts of individual components were calculated based on the relative % peak areas (FID response),
without using correction factor.

Antibacterial and antifungal activity against human pathogens

The antibacterial and antifungal activity of the essential oil was determined using disc diffusion and
microdilution broth assays as previously described (Chaturvedi et al., 2018). The bacterial strains were
MRSA {methicillin-resistant Staphylococcus aureus (33591)}, MRSE {methicillin resistant
Staphylococcus epidermidis (51625)}, MRSA {methicillin-resistant Staphylococcus aureus (BAA-44)},

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Staphylococcus aureus (MTCC-96), Staphylococcus epidermidis (MTCC-435), and Mycobacterium
smegmatis (UDSC-MC2 155). The fungal strains were Candida tropicalis (ATCC-2013180), Candida
albicans (ATCC-14053), Candida krusei (ATCC-14243), Candida kefyr (ATCC-204093) and Candida
albicans (clinical isolate). The activities were measured in terms of the net zone of inhibition (ZOI) and
minimum inhibitory concentration (MIC). The experiments were performed in triplicate. The bacterial
strains were obtained from the Microbial Type Culture Collection Centre (MTCC), Institute of Microbial
Technology (IMT) Chandigarh, India.

Antifungal activity against phyto-pathogens: poison food technique (contact assay)

The antifungal activity of the essential oil was evaluated against three phytopathogenic fungi, Curvularia
lunata (CIMAP: SB-82013), Rhizoctonia solani (CIMAP: Cor-Rh1) and Fusarium chlamydosporum
(CIMAP: La-72016A) by contact assay on Potato Dextrose Agar (PDA) medium as described previously
(Saroj et. al., 2015). The test plant pathogenic fungi were obtained from CSIR-Central Institute of
Medicinal and Aromatic Plants, Plant Pathology Department. The plates were prepared by adding
different concentrations of the essential oil (0.2 %, 0.5 %, and 1.0 %) into 10 mL of the PDA medium.
The medium was poured into Petri plate at 40–450C. To ensure proper mixing of the essential oil, 0.05 %
tween-80 was added. A 6 mm disc of three test fungi (four days old culture) was placed on the PDA filled
Petri plates. All Petri plates were incubated at 25 ± 2°C. The percentage of inhibition colony diameter was
measured day by day. All tests were carried out in triplicate. Plates without essential oil served as a
control. Percentage of the growth inhibition was calculated as follows:

References
Adams, R.P. 2007. Identification of essential oil components by gas chromatography/mass spectrometry.
Allured Publishing Corp., Carol Stream, Illinois, USA.
Chaturvedi, T., Kumar, A., Kumar, A., Verma, R.S., Padalia, R.C., Sundaresan, V., Chauhan, A., Saikia,
D., Singh, V.R., Venkatesha, K.T. 2018. Chemical composition, genetic diversity, antibacterial,
antifungal and antioxidant activities of camphor-basil (Ocimum kilimandscharicum Guerke), Ind.
Crops Prod. 118, 246-258.
Saroj, A., Pragadheesh, V.S., Yadav, A., Singh, S.C., Samad, A., Negi, A.S., Chanotiya, C.S. 2015. Anti-
phytopathogenic activity of Syzygium cumini essential oil, hydrocarbon fractions and its novel
constituents. Ind. Crops Prod. 74, 327-335.

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Table S1-Essential oil composition of the senescent leaves of Psidium guajava
S. no. Compound RIa RIb Content (%)

1. α-Pinene 934 932 0.8

2. Benzaldehyde 959 952 0.6
3. β-Pinene 981 974 0.2
4. 6-Methyl-5-hepten-2-one 985 981 t
5. Myrcene 991 988 0.4
6. α-Phellandrene 1007 1002 0.1
7. p-Cymene 1025 1020 0.2
8. Limonene 1029 1024 29.1
9. 1,8-Cineole 1035 1026 2.6
10. (Z)-β-Ocimene 1040 1032 t
11. (E)-β-Ocimene 1048 1044 0.1
12. γ-Terpinene 1060 1054 0.1
13. Terpinolene 1092 1086 t
14. Linalool 1097 1095 t
15. Terpinen-4-ol 1181 1174 0.1
16. α-Terpineol 1191 1186 0.5
17. cis-Carveol 1231 1226 0.4
18. Neryl acetate 1365 1359 0.1
19. α-Copaene 1383 1373 2.3
20. (E)-Caryophyllene 1428 1417 15.7
21. α-Humulene 1462 1452 2.0
22. allo-Aromadendrene 1468 1458 0.3
23. γ-Muurolene 1485 1478 0.6
24. Germacrene D 1488 1480 0.1
25. Viridiflorene 1495 1496 1.2
26. α-Selinene 1504 1498 1.3
27. α-Muurolene 1506 1500 t
28. (Z)-α-Bisabolene 1514 1506 t
29. β-Bisabolene 1515 1505 0.3
30. γ-Cadinene 1524 1513 0.2

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31. δ-Cadinene 1530 1522 1.7
32. trans-Cadina-1,4-diene 1540 1533 0.5
33. α-Calacorene 1549 1544 0.1
34. (E)-Nerolidol 1567 1561 4.0
35. Caryophyllenyl alcohol 1584 1570 0.3
36. Caryophyllene oxide 1594 1582 8.8
37. Gleenol 1599 1586 0.1
38. Ledol 1612 1602 0.2
39. Humulene epoxide II 1615 1608 1.3
40. 1-epi-Cubenol 1632 1627 0.2
41. Muurola-4,10(14)-dien-1-β-ol 1637 1630 2.5
42. Caryophylla-4(12),8(13)-dien-5-ol 1646 1639 6.5
43. epi-α-Muurolol 1648 1640 t
44. α-Muurolol 1652 1644 1.1
45. α-Cadinol 1662 1652 3.4
46. (2Z,6E)-Farnesol 1727 1722 0.9
Class composition
Monoterpene hydrocarbons 30.8
Oxygenated monoterpenes 3.7
Sesquiterpene hydrocarbons 26.3
Oxygenated sesquiterpenes 29.3
Others 0.8
Total identified (%) 90.9

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Table S2: Antimicrobial activity of the senescent leaves of Psidium guajava
Strain ZOI (mm) MIC (mg/mL)

Bacterial strain
Staphylococcus aureus (MRSA) 03 0.065
Staphylococcus aureus (BAA-44) 05 0.065
Staphylococcus epidermidis (MRSE) 03 0.065
Staphylococcus aureus (MTCC-96) 08 0.261
Staphylococcus epidermidis (MTCC-435) 08 0.130
Mycobacterium smegmatis (UDSC-MC2 155) 06 0.261
Fungal strain
Candida tropicalis (ATCC 2013180) 02 >33.42
Candida albicans (ATCC-14053) 02 >33.42
Candida krusei (ATCC14243) 03 16.71
Candida kefyr (ATCC-204093) 02 >33.42
Candida albicans (clinical isolate) 02 >33.42

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Figure S1: Total ion chromatogram of the essential oil of senescent leaves of Psidium guajava

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Figure S2: Percentage of radial growth inhibited by essential oil of Psidium guajava against three
phytopathogenic fungi at 0.2 %, 0.5 %, and 1.0 % after 5 days incubation at 25±2°C (average data of five
days). Cl: Curvularia lunata, Rs: Rhizoctonia solani; Fc: Fusarium chlamydosporum

Figure S3: Radial growth inhibition of fungi with 1% essential oil of Psidium guajava (5 days old
culture).
A: control; B: growth inhibition of Curvularia lunata;
C: control; D: growth inhibition of Rhizoctonia solani;
E: control; F: growth inhibition of Fusarium chlamydosporum