Nutritional and nutraceutical comparison of Jamaican Psidium cattleianum (strawberry guava) and Psidium guajava (common guava) fruits

Food Chemistry 134 (2012) 1069–1073

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Nutritional and nutraceutical comparison of Jamaican Psidium cattleianum (strawberry guava) and Psidium guajava (common guava) fruits Kayanne P. McCook-Russell a, Muraleedharan G. Nair b, Petrea C. Facey a, Camille S. Bowen-Forbes a,⇑ a b

Department of Chemistry, The University of the West Indies, Mona, Kingston 7, Jamaica Bioactive Natural Products and Phytoceuticals Laboratory, Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA

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Article history: Received 6 April 2011 Received in revised form 27 February 2012 Accepted 6 March 2012 Available online 16 March 2012 Keywords: Psidium cattleianum Psidium guajava Strawberry guava Antioxidant COX enzyme Jamaica

a b s t r a c t Psidium cattleianum (strawberry guava) is one of many underutilised edible fruits that grow wild in Jamaica, and could potentially be commercially exploited to yield health and economic benefits. In this study, the total phenolics, proximate contents, and antioxidant, anti-inflammatory, and antimicrobial activities of P. cattleianum and P. guajava (common guava), a well-known species, were compared. Strawberry guavas were found to be superior to common guavas in antioxidant and antimicrobial activities, total phenolics and vitamin C content. They also possessed relatively high fibre content (24.9%). The hexane and ethyl acetate extracts of strawberry guavas showed cyclooxygenase-2 enzyme inhibitory activities of 18.3% and 26.5%, respectively (250 lg/mL), indicating anti-inflammatory activity. The EtOAc and MeOH extracts of P. guajava showed 56.4% (COX-2) and 44.1% (COX-1) inhibitory activity, respectively. Additionally, nine compounds were isolated from strawberry guava fruits, some of which demonstrated anti-inflammatory activity. These results indicate that strawberry guavas are beneficial for health. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The Myrtaceae family consists of at least 133 genera and more than 3800 species (Wilson, O’Brien, & Gadek, 2001). Many fruits of this family have a history of being used in traditional medicines in ethnobotanical practices in the tropical and subtropical regions (Marin et al., 2008). Members of the Myrtaceae family include the Eugenia, Myrcianthes, Campomanesia and Psidium genera (Marin et al., 2008). The Psidium genus is represented by approximately 120–150 species and may be found throughout the tropics and subtropics of America and Australia (Pino, Bello, Urquiola, Marbot, & Martí, 2004). The constituents of some of the species have been studied, with special focus being placed on the flavour components of the fruits. Antioxidant activities of some members have also been examined. The most popular Psidium species, guajava, simply called guava, has been extensively examined. The round-oval fruit is green-yellow and shows a light yellow, pink (Shreier & Idstein, 1985), white or salmon pulp depending on the variety (Mercadante, Steck, & Pfander, 1999). It grows on a low evergreen tree or shrub and can attain a height between 6 and 33 feet (2–11 m). The trees grow rapidly and fruit within 2–4 years from the seed. World production of guavas is estimated at approximately 500,000 metric tonnes, with Brazil, Colombia, Mexico and Venezuela producing significant quantities from the South American re⇑ Corresponding author. Tel.: +1 876 927 1910; fax: +1 876 977 1835. E-mail address: [email protected] (C.S. Bowen-Forbes). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.03.018

gions. The industry provides a range of processed products such as beverages, syrup, ice cream, jams and jellies, to name a few (Jiménez-Escrig, Rincón, Pulido, & Saura-Calixto, 2001). The major producers of guava products are South Africa, India, Hawaii, Colombia, Puerto Rico, Jamaica, Brazil and Israel (Mercadante et al., 1999). In Jamaica, P. guajava is commonly used in the manufacture of jams, jellies, ice cream and juices, among other products. Psidium cattleianum Sabine (strawberry guava) is often described as being more aromatic than Psidium guajava (common guava). There are two known cultivars – the red fruit and the yellow fruit (Luximon-Ramma, Bahorun, & Crozier, 2003). This species is a shrub or tree which can grow 3–10 m high and is very common to Southern Brazil (Marin et al., 2008). Other names synonymous with strawberry guava include Psidium littorale Raddi or purple guava. The fruit is widely cultivated in Central and South America and has been naturalised in Jamaica in the parishes of Clarendon, Manchester and St. Ann (Adams, 1972). In Hawaii, the plant is actually considered as a forest weed and is able to propagate quite easily from the seed. It has not been as extensively studied as the guajava species, however the volatile constituents of the fruit have been quantified and characterised (Pino, Marbot, & Vázquez, 2001). Additionally, P. cattleianum, P. guajava and other Mauritia grown fruits have been analysed for their antioxidant capacity, total polyphenolic and nutritional contents. This research was undertaken to compare the nutraceutical and nutritional value of the P. cattleianum to the more well-known P. guajava (Luximon-Ramma et al.,

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2003). In the present study, the phytochemistry of P. cattleianum fruits, which has not been previously reported, was investigated. The anti-inflammatory activity of the fruits of this species is now being reported for the first time. An exploration of the natural products, antioxidant, antimicrobial and anti-inflammatory properties of Jamaican strawberry guavas would shed light on this essentially unknown fruit, which could become a normal part of people’s diets, and positively impact health. Additionally, the fruit could be potentially exploited to improve the country’s economy via commercialisation. 2. Materials and methods 2.1. General experimental procedures The 1H and 13C NMR spectra were recorded in deuterated chloroform, acetone or methanol using Bruker Avance 200 and 500 MHz NMR spectrometers (Bruker, Karlsruhe, Germany). Silica gel (230–400 mesh) was used for column chromatography. Folin– Ciocalteau phenol reagent, gallic acid, 20 ,20 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma Aldrich Inc., St. Louis, MO, USA. The bacterial cultures used in the antimicrobial assay were provided by the University Hospital of the West Indies (UHWI), St. Andrew, Jamaica. The COX-1 enzyme was prepared from ram seminal vesicles purchased from Oxford Biomedical Research, Inc. (Oxford, MI, USA). COX-2 enzyme was prepared from Spodoptera frugiperda cells cloned with human PGHS-2 (prostaglandin endoperoxide H synthase-2) enzyme. P. cattleianum fruits (orange-red) were harvested from Mason River, Clarendon, while Psidium guajava fruits (pale pink) was collected from Papine, St. Andrew, both in Jamaica, in October and November, 2009, respectively. A portion of the hexane extract was separated using an Agilent GC 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with an SLBTM-5MS fused silica capillary column (30 m  0.25 m  0.25 lm, Supelco, Bellefonte, PA, USA) with the GC oven temperature programme: 60 °C (50 ) to 300 °C at 10 °C/min. Splitless injection was used at a temperature of 250 °C and He carrier gas at 1.3 mL/min flow. The model 5973 mass-selective detector was operated at a scanning mass range of 50–600 m/z with 70 eV ionisation energy, 230 °C EI ion source temperature and the quadrupolar mass detector at 150 °C. 2.2. Extraction of fruits Fresh, fully ripe fruits of P. cattleianum (2129 g) and P. guajava (366 g) were lyophilised to yield 443 and 63 g of dried material, respectively. A portion of the former (413 g) was extracted successively with hexane (2  3L), ethyl acetate (2  3L) and methanol (2  3L). Smaller volumes (2  600 mL) were used for extracting P. guajava (63 g). The first extraction was done for 3 h and the other was carried out for 16 h. An orbital shaker was used to facilitate extraction. The extracted plant material were filtered, pooled as appropriate, and concentrated en vacuo to give 5.9, 2.4 and 220 g hexane, ethyl acetate and methanol extracts, respectively, for P. cattleianum and 0.83, 1.2 and 24 g, respectively, for P. guajava.

which were obtained during elution with 15:85, ethyl acetate/hexane. A portion of the hexane extract was analysed by GC/MS. The same method of purification was applied to the ethyl acetate extract (2.2 g). This yielded compound 1 (27 mg), a mixture of compounds 3 and 4 (10.3 mg), a mixture of compounds 5 and 6 (8.3 mg) and compound 7 (107 mg). Compounds 1–6 were isolated during elution with the same solvent system as that in the hexane extract while 7 was obtained using a solvent system of ethyl acetate/hexane (60:40, v/v). A portion of the methanolic extract (77 g) was dissolved in water (500 mL) and extracted with ethyl acetate (4 x 400 mL). The resultant concentrate was subjected to column chromatography using silica gel. Fraction A was obtained by eluting the column with ethyl acetate/hexane (50:50, v/v), while fraction B contained compounds which were eluted in EtOAc. Successive column chromatography of fraction A yielded compounds 1 (14.3 mg) and 2 (15 mg), both isolated using ethyl acetate/hexane (2:98, v/v), while 7 (53.8 mg) and a mixture of 8 and 9 (92 mg) were isolated from fraction B during elution with solvent systems comprising of 60:40 (v/v) and 50:50 (v/v) ethyl acetate/hexane, respectively. Compounds 8 and 9 were present in an approximate mole ratio of 1:1, based on the intensities of the NMR signals of the respective compounds. The structures of the compounds are shown in Fig. 1. 2.4. Antioxidant assay The antioxidant activities of the extracts were determined using Trolox equivalent antioxidant capacity (TEAC) (Seeram et al., 2006). 20 ,20 -Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt radical cations were generated by adding solid manganese dioxide to a 5 mM aqueous solution of ABTS. A calibration curve was established using ethanolic solutions of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The methanolic extracts of the fruits were tested at a concentration of 1 mg/100 lL. For each measurement, ABTS+ solution (2 mL) was added to each sample and standard (20 lL). Samples were assayed in triplicate and the absorbance was read at 734 nm. TEAC values were calculated and expressed as Trolox equivalents (lmol/g). 2.5. Total phenolic content Samples were analysed spectrophotometrically for total phenolic contents using 10% Folin–Ciocalteau reagent, and gallic acid was used as a reference. Standards of concentration 0.8, 1.6, 2.4, 3.2 and 4.0 ppm were prepared from a stock solution of gallic acid with a

H

R H

HO H

H

H

HO

O

H

R H

Silica gel column chromatography was carried out on a portion of the hexane extract (4.2 g) using increasing concentrations of EtOAc/hexane. This led to the isolation of compounds 1 and 2 (3.25 g and 50.7 mg). Further purification of a mixed fraction from this extract produced a mixture of compounds 3 and 4 (17 mg),

HO

H

O OH

HO

2.3. Isolation of natural products from P. cattleianum

COOH

H

H

O

COOH

R1 R

O

O

Fig. 1. Compounds 2–6 and 8–9.

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concentration of 100 ppm. The samples (1.2 – 1.5 g) were extracted with boiling water (100 mL) for 4 min, then filtered and made up to 100 mL. A portion of this solution (1 mL) was diluted to 25.0 mL and the procedure followed as that for the standards. Absorbances were read at 750 nm and values calculated and expressed as lg gallic acid equivalents (GAE)/g fresh weight (Chen & Yen, 2006). 2.6. Antimicrobial assay Antibacterial assay was carried out using the hexane, ethyl acetate and methanolic extracts of P. cattleianum, by employing the disc diffusion assay (Bauer, Kirby, Sherris, & Turck, 1966; Boyd, 1984). The compounds obtained from the ethyl acetate extract were also analysed. The bacteria used in the assay were Bacillus subtilis, Pseudomonas aureginosa, Escherichia coli and Staphylococcus aureus. For the crude extracts, 500 lg/disc was used while 50 lg/ disc was used for individual compounds. The activities of the extracts and compounds were compared to that of the known antibiotic, gentamicin, at a concentration of 10 lg/disc. 2.7. COX enzyme inhibitory assay The method used for the COX enzyme inhibitory assay was previously reported (Bowen-Forbes, Mulabagal, & Nair, 2009). Extracts and pure compounds were tested at 250 and 25 mg/L respectively, using dimethyl sulphoxide (DMSO) as the solvent. Due to paucity of COX enzymes, the analyses were done in duplicate. 2.8. Proximate analysis The fat, protein, ash, calcium and crude fibre contents of the Psidium species were analysed using AOAC (2005) methods. Vitamin C was determined using an iodometric titration (Volpe, Collins, Simoni, & Silva, 1999). 3. Results and discussion 3.1. Characterisation of compounds The identities of all compounds were elucidated by NMR spectral methods. The metabolites were identified as triolein (1), bsitosterol (2) (Nes, Norton, & Benson, 1992), ursolic acid (3) (Mahato & Kundu, 1994), oleanolic acid (4) (Mahato & Kundu, 1994), 2ahydroxyursolic acid (5) (Begum et al., 2002), 2a-hydroxyoleanolic acid (6) (Abou-Mansour et al., 2005), citric acid (7) (Pouchert & Behnke a, 1993), and a mixture of citrate esters, 1,2,3-propanetricarboxylic acid-2-hydroxy-1-methyl ester (8) (Han et al., 2001) and 1,2,3-propanetricarboxylic acid-2-hydroxy-2-methyl ester (9) (Jayaprakasha et al., 2007). In addition to using NMR spectroscopy, the identities of the compounds triolein, b-sitosterol, ursolic acid, oleanolic acid, and citric acid, were further confirmed based on TLC comparison with authentic samples. Additionally, GC/MS analyses confirmed the presence of triolein and revealed that caryophyllene and its oxide were also present. Triolein was the major component of the hexane extract with an abundance of 76%, representing 0.2% of the fruit. b-Sitosterol is a common plant sterol which has been associated with the Psidium genus. Ursolic and oleanolic acids, which were isolated from both the hexane and ethyl acetate extracts, were obtained as a mixture. Both acids have been found in the fruit of P. guajava (Peng, Hsieh, & Chen, 2008) and have been shown to exhibit anti-inflammatory properties in laboratory animals (Liu, 1995). Citric acid, which was relatively abundant in the fruit, comprised 5% of the ethyl acetate extract. This compound is prevalent in citrus fruits and is associated with the sour taste of limes and lemons.

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The 1H NMR data for the citrate ester showed two tall singlets at 3.68 (9) and 3.77 (8) ppm which are characteristic of protons belonging to a methoxy group. There was also a broad singlet at 5.10 ppm, indicative of an exchangeable proton belonging to an amine, acid or alcohol. This corresponded to the unmethylated carboxyl groups and the hydroxyl moiety. The 13C NMR data showed three methlyene carbons at 43.8 (9), 44.1 (8) and 44.2 ppm (9), all of which were in the region of the chemical shift for the methylene groups in citric acid. Additionally, there were two signals resonating at 52.3 (8) and 53.2 ppm (9), confirming the presence of methoxy groups. A pair of oxygenated quaternary carbon atoms was also observed in the 13C NMR spectrum at 72.8 (8) and 73.2 ppm (9). A cluster of peaks in the region of 170–176 ppm indicated that there were carboxylic and carboxylate groups in different chemical environments. These NMR data suggested that a mixture of citric acid methyl esters was present. They were determined to be 1,2,3-propanetricarboxylic acid-2-hydroxy-1-methyl ester (8) and 1,2,3-propanetricarboxylic acid-2-hydroxy-2-methyl ester (9) based on comparison with literature data. Citric acid (7) was a major natural product found in the P. cattleianum fruits. Considering that compounds 8 and 9 were obtained from the methanol extract, the possibility exists that were derived from methylation of citric acid during extraction. Caryophyllene and its epoxide are well-known volatile constituents of Psidium cattleainum. Previous studies on the volatile flavour components of the fruit using GC/MS showed a concentration of 10.25 ppm for b-caryophyllene and 1.16 ppm for its epoxide. Both are believed to contribute a spicy note to the flavour of the fruit (Pino et al., 2001). 3.2. Antioxidant assay The methanolic extracts were analysed for antioxidant activity. The antioxidant components of the fruit are expected to be concentrated in this extract. The TEAC of strawberry guavas was found to be three times that of P. guajava (i.e. 11.3 + 0.1 compared to 3.8 + 0 lmol Trolox/g fresh weight). In a previous study carried out on Mauritian fruits, red strawberry guavas were found to possess almost seven times the Trolox equivalence as that of common guavas (Luximon-Ramma et al., 2003). The levels reported were, however, significantly higher than the ones now being reported. The dissimilarity between the results is likely to be due to differences in extraction protocols as well as environmental factors. None of the metabolites which were isolated from strawberry guava showed antioxidant activity at the concentration tested. 3.3. Total phenolic content The total phenolic contents within the cattleianum and guajava species was 4439 and 1952 lg GAE/g fresh weight, respectively. In other words, strawberry guavas had more than twice as much polyphenols as common guavas. Both red and yellow varieties of P. cattleianum from Mauritia showed phenolic contents exceeding 5000 lg GAE/g fresh weight. The levels reported in pink and white Mauritian common guavas were comparatively lower with values of 1264 + 60 and 2473 + 45 lg GAE/g fresh weight, respectively (Luximon-Ramma et al., 2003). 3.4. Antimicrobial activity The methanolic extract of P. cattleianum possessed intermediate activity against B. subtilis and S. aureus. No other extract produced positive results against the bacteria tested. No activity was demonstrated by the P. guajava fruit extracts. S. aureus is a Gram-positive bacterium responsible for diseases such as pneumonia and meningitis while the less harmful B. subtilis is not a human pathogen. The

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essential oils of the leaves of strawberry guava showed 55 and 58% inhibition of S. aureus and P. aeruginosa, respectively (Apel et al., 2006), while in folk medicine, extracts of the roots, bark and leaves of P. guajava have been used to treat gastroenteritis, vomiting and diarrhoea (Peng et al., 2008). 3.5. COX-1 and -2 enzyme inhibitory assay The cyclooxygenase enzyme catalyses the oxidation of arachidonic acid to prostaglandins and thromboxanes. The isoforms of the enzyme COX-1 and COX-2 can be inhibited by non-steroidal anti-inflammatory agents, resulting in reduction in inflammation and pain relief. Inhibition of the COX-2 isoform is linked to a reduction in malignancy (Bowen-Forbes et al., 2009). When tested at a concentration of 250 lg/mL, the hexane and ethyl acetate extracts of P. cattleianum showed respective COX-2 activities of 18.3 and 26.5%. The mixture of ursolic and oleanolic acids (3 and 4) showed 19.4% inhibition of the COX-2 enzyme at 250 lg/mL. The highest overall activity was exhibited by the mixture of their corresponding 2a-hydroxy analogues (5 and 6), which demonstrated 52.9 and 43.1% inhibition against the COX-2 and COX-1 enzymes, respectively. The ethyl acetate extract of P. guajava showed notable activity (56.4%) against the COX-2 isoform, while the methanolic extract exhibited 44.1% inhibition against the COX-1 enzyme (Fig. 2). 3.6. Proximate analysis The results in Table 1 indicate that P. cattleianum is higher in fat, calcium and vitamin C than its more common relative, P. guajava. Both species had low fat contents, comparable to that noted by Popenoe, 1948 (1.0%). The average % protein for strawberry guava was approximately four times greater than the 0.53% calculated for P. littorale Radii var. lucidum (yellow Cattley guava) (Hall, Smoot, Knight & Nagy, 1980). In this case, crude fibre was quantified with common guava having the greater fibre content. Previous studies have shown that the peel and pulp of P. guajava possess high levels of dietary fibre with associated natural antioxidant components (Jiménez-Escrig et al., 2001). It can therefore be classified as a fruit

Table 1 Proximate analysis, total phenolics, and antioxidant activity (TEAC) of P. cattleianum and P. guajava fruits.a

% Fat % Protein % Ash % Crude fibre Calcium Vitamin C Total polyphenols/lg GAE TEAC/lmol TE

P. cattleianum

P. guajava

1.24 + 0.01 2.10 + 0.03 3.05 + 0.01 324.9 + 1.7 63.2 + 0.7 2091 + 42 4439 + 159 11.3 + 0.1

0.97 + 0.04 4.22 + 0.11 3.12 + 0.03 30.9 + 2.8 29.8 + 2.0 1200 + 46 1952 + 111 3.8 + 0.0

TE = Trolox equivalent (lmol Trolox/g fresh weight), GAE = gallic acid equivalents (lg GA/g fresh weight). a Vitamin C and calcium contents are expressed in lg g1 fresh weight.

rich in antioxidant dietary fibre (AODF), and polyphenolic compounds (Saura-Calixto, 1998). Crude fibre does not encompass the total fibre composition of a food and so the possibility exists that P. cattleianum is also high in dietary fibre and may, by extension, be classified as an AODF. The vitamin C content was much greater in Jamaican strawberry guava than that noticed in the red variety found in Mauritia, which had a value of 242 + 15 lg/g fresh weight. The value we obtained for P. guajava (1200 lg/g fresh weight) was comparable to that found in the Mauritian white common guavas (1426 fresh weight) (Luximon-Ramma et al., 2003). The Jamaican P. guajava species exhibited higher fat, protein, ash and crude fibre contents (see Table 1) compared to those quoted for the variety from Venezuela (0.4–0.7, 0.8–1.5, 0.5–1.0, and 2.0–7.2%, respectively) (Jiménez-Escrig et al., 2001). The general composition of a fruit depends on its stage of maturity (Chyau Chen & Wu, 1992), the season and where it is grown. This may partially account for the differences in the parameters tested by different researchers. 3.7. Conclusion For the most part, P. cattleianum was found to be a better source of nutrients than P. guajava. It showed greater antioxidant and

a

b

Fig. 2. Cyclooxygenase 2 enzyme inhibitory activities of positive controls, Aspirin, CelebrexTM and VioxxÒ, at 60 lM, 26 and 32 nM, respectively (a) and extracts and isolated h i rate of O2 uptake of COX sample solution compounds tested at 250 and 25 lg/mL, respectively (b). Vertical bars represent ± SD for each data point (n = 2). % Inhibition ¼ 1  initial  100%. initial rate of O2 uptake of COX DMSO solution

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antimicrobial activities, as well as higher vitamin C content. Its total phenolic content was almost twice that of P. guajava, which is advantageous, as these chemicals aid with free radical scavenging. Psidium guajava however does exhibit greater anti-inflammatory activity than P. cattleianum. This research has demonstrated the great potential of Jamaican strawberry guavas. Based on the present findings, the fruit may be made more popular due to its health beneficial properties, as well as the potential for commercial production of value-added products. Commercialisation would be facilitated by large scale cultivation, which would be advantageous to the country’s agricultural sector. More detailed study of other parts of the plant to further explore its nutraceutical potential will be undertaken. Acknowledgements We gratefully acknowledge funding in the form of a New Initiative Grant and a Research Fellowship received from The University of the West Indies, Mona Campus, for the undertaking of this project. References Abou-Mansour, E., Djoukeng, Tabacchi R., Tapondjou, A. L., Bouda, H., Lontsi, D., & Djoukeng, Tabacchi. (2005). Antibacterial triterpenes from Syzygium guineense (Myrtaceae). Journal of Ethnopharmacology, 101, 283–286. Adams, C.D. (1972). Flowering Plants of Jamaica, (p. 519). Glasgow, Scotland: Robert MacLehose and Company Ltd. AOAC (2005). Official Methods of Analysis (18th ed.). Gaithersburg, MD: AOAC International. Apel, M. A., Lima, M. E. L., Souza, A., Cordeiro, I., Young, M. C. M., Sobral, M. E. G., et al. (2006). Screening of the biological activity from essential oils of native species from the Atlantic rain forest (São Paulo – Brazil). Pharmacology online, 3, 376–383. Bauer, A. W., Kirby, W. M. M., Sherris, J. C., & Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45, 493–496. Begum, S., Sultana, I., Siddiqui, B. S., Shaheen, F., & Gilani, A. H. (2002). Structure and spasmolytic activity of eucalyptanoic acid from Eucalyptus camaldulensis var. obtusa and synthesis of Its active derivative from oleanolic acid. Journal of Natural Products, 65, 1939–1941. Bowen-Forbes, C. S., Mulabagal, V., & Nair, M. G. (2009). Ursolic acid analogues: non-phenolic functional food components in Jamaican raspberry fruits. Food Chemistry, 116, 633–637. Boyd, R. F. (1984). General Microbiology, p. 608. Times Mirror/Mosby College, U.S.A. Chen, H., & Yen, G. (2006). Antioxidant activity and free radical-scavenging capacity of extracts from guava (Psidium guajava L.) leaves. Food Chemistry, 101, 686–694. Chyau, C., Chen, S., & Wu, C. (1992). Differences of Volatile and Nonvolatile Constituents between Mature and Ripe Guava (Psidium guajava Linn) Fruits. Journal of Agricultural and Food Chemistry, 40, 846–849.

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