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Health Effects of Psidium guajava L. Leaves: An Overview of the Last Decade

Elixabet díaz-de-cerio.

1 Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd. Fuentenueva s/n, 18071 Granada, Spain; [email protected] (E.D.-d.-C.); [email protected] (A.F.-G.); [email protected] (A.S.-C.)

2 Functional Food Research and Development Center, Health Science Technological Park, Avd. Del Conocimiento, Bioregion Building, 18100 Granada, Spain

Vito Verardo

3 Department of Nutrition and Food Science, University of Granada, Campus of Cartuja, 18071 Granada, Spain; [email protected]

Ana María Gómez-Caravaca

Alberto fernández-gutiérrez, antonio segura-carretero.

Today, there is increasing interest in discovering new bioactive compounds derived from ethnomedicine. Preparations of guava ( Psidium guajava L.) leaves have traditionally been used to manage several diseases. The pharmacological research in vitro as well as in vivo has been widely used to demonstrate the potential of the extracts from the leaves for the co-treatment of different ailments with high prevalence worldwide, upholding the traditional medicine in cases such as diabetes mellitus, cardiovascular diseases, cancer, and parasitic infections. Moreover, the biological activity has been attributed to the bioactive composition of the leaves, to some specific phytochemical subclasses, or even to individual compounds. Phenolic compounds in guava leaves have been credited with regulating blood-glucose levels. Thus, the aim of the present review was to compile results from in vitro and in vivo studies carried out with guava leaves over the last decade, relating the effects to their clinical applications in order to focus further research for finding individual bioactive compounds. Some food applications (guava tea and supplementary feed for aquaculture) and some clinical, in vitro, and in vivo outcomes are also included.

1. Introduction

Ethnomedicine, which refers to the study of traditional medical practice, is an integral part of the culture and the interpretation of health by indigenous populations in many parts of the world [ 1 ]. For example, Indian Ayurveda and traditional Chinese medicine are among the most enduring folk medicines still practiced. These systems try to promote health and improve the quality of life, with therapies based on the use of indigenous drugs of natural origin [ 2 ]. Given that plants have been widely used as herbal medicines, several approaches are now being carried out to discover new bioactive compounds [ 3 ].

Psidium guajava L., popularly known as guava, is a small tree belonging to the myrtle family (Myrtaceae) [ 4 ]. Native to tropical areas from southern Mexico to northern South America, guava trees have been grown by many other countries having tropical and subtropical climates, thus allowing production around the world [ 5 ]. Traditionally, preparations of the leaves have been used in folk medicine in several countries, mainly as anti-diarrheal remedy [ 6 ]. Moreover, other several uses have been described elsewhere on all continents, with the exception of Europe [ 6 , 7 , 8 ]. Figure 1 summarizes the main traditional uses of guava leaves in the main producer countries. Depending upon the illness, the application of the remedy is either oral or topical. The consumption of decoction, infusion, and boiled preparations is the most common way to overcome several disorders, such as rheumatism, diarrhea, diabetes mellitus, and cough, in India, China, Pakistan, and Bangladesh [ 6 , 7 , 8 , 9 ], while in Southeast Asia the decoction is used as gargle for mouth ulcers [ 6 , 8 , 9 ] and as anti-bactericidal in Nigeria [ 8 , 9 ]. For skin and wound applications, poultice is externally used in Mexico, Brazil, Philippines, and Nigeria [ 6 , 7 , 8 , 9 ]. In addition, chewing stick is used for oral care in Nigeria [ 9 ].

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Main traditional uses of guava leaves in the principal producer countries.

Currently, there is increasing interest in studying of plants regarding their chemical components of bioactive compounds, their effects on several diseases, and their use for human health as functional foods and/or nutraceuticals [ 10 ]. In recent years, guava leaves tea and some complementary guava products are available in several shops in Japan as well as on the Internet [ 11 ], because guava leaf phenolic compounds have been claimed to be food for specified health use (FOSHU), since they have beneficial health effects related to the modulation of blood–sugar level [ 12 ]. Thus, the aim of this review was to summarize the biological activities, in vitro and in vivo, studied in the last decade on P. guajava L. leaves, relating them to the international classification of diseases provided by the World Health Organization. In addition, the beneficial effects of some applications of guava leaves are also been examined. For this purpose, a comprehensive review of the literature from 2004 to 2016 was done, although more recent studies have also been included. Reviewed journals, websites, books, and several databases as “Scopus”, “Google Scholar”, “PubMed”, and “ScienceDirect”, were used to compile them. To ensure that relevant works are included, terms such as “ Psidium guajava ”, “guava”, “leaves”, “in vitro”, “in vivo”, “clinical”, “trial”, “food application”, and those related with the diseases such as “bacteria”, “cancer”, “blood”, “glycaemia”, and “oral”, among others were matched in the search. Only complete available works published in English, Spanish, and Portuguese have been included.

2. Pharmacological Properties

2.1. in vitro studies, 2.1.1. infectious and parasitic diseases.

Aqueous and organic extracts of guava leaves have been demonstrated to have antibacterial activity due to an inhibitory effect against antibiotics-resistant clinical isolates of Staphylococcus aureus strains [ 13 , 14 ]. Despite using the same diffusion method, differences are noticed in their inhibition zones, as shown in Table 1 , probably due to extraction method or the dose assayed. A methanol extract exerted antibacterial effects, preventing the growth of different strains from several bacteria such as Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa , Proteus spp., and Shigella spp. [ 15 ]. Furthermore, different extracts of the leaves such as aqueous, acetone–water, methanolic, spray-dried extracts, and the essential oil, showed potential inhibitory activity against Gram-positive and Gram-negative bacteria and fungi [ 16 , 17 , 18 , 19 , 20 ]. In these works, it is noticeable that Gram-positive bacteria exhibited greater inhibition zones and minimum inhibitory concentrations (MICs) than Gram-negative. Concerning the anti-fungal activity, less inhibition than bacteria is reported [ 16 , 17 ], except for Candida krusei and Candida glabrata which provided higher inhibition [ 18 ], and for Aspergillus spp. for which no activity was found [ 16 ] ( Table 1 ). Moreover, Bezerra et al. [ 21 ] evaluated the effect of guava leaves on different bacterial strains, concluding that the synergistic action between the leaves and various antibiotics boosted its anti-bacterial activity. This effect was also observed by Betoni et al. [ 22 ] with target drugs for the protein synthesis, cell-wall synthesis, and folic acid. However, the latter did not find synergic effect with gentamicin, perhaps because the time of maceration was lower than the time used by Bezerra et al. [ 21 ], and also the solvent was different ( Table 1 ).

In vitro assays against infectious and parasitic diseases.

Acetone (Ac); N , N -dimethylformamide (DMF); dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); effective concentration (EC 50 ); inhibition zone (iz); inhibitory concentration (IC 50 ); lethal concentration (LC 50 ); minimum bactericidal concentration (MBC); minimum fungicidal concentration (MFC); minimum inhibitory concentration (MIC); total flavonoid content (TFC); total phenolic content (TPC); Tetrazolium (MTT); ↑ increases the affect; ↓ decreases the effect.

Metwally et al. [ 23 ] associated the antimicrobial activity against some bacteria and fungi with five flavonoids isolated from the leaves. This effect was also related to the concentration of tannins in the leaves [ 24 ] and to the content of gallic acid and catechin [ 19 ]. Additionally, the activity against bacterial and fungal pathogens was traced to betulinic acid and lupeol [ 25 ]. In fact, these works are focused on the activity of these compounds, rather than on the effect of the whole extract of the leaves.

In addition, leaf acetone extract of P. guajava has also exhibited moderate acaricidal and insecticidal activities causing the dead of Hippobosca maculata adult fly [ 26 ].

Furthermore, Adeyemi et al. [ 27 ] suggested that an ethanol extract from the leaves function as a trypanocide agent, since its inhibition of Trypanosoma brucei brucei growth proved similar to that of the reference drugs. Kaushik et al. [ 28 ] proposed the leaves as an anti-malaria agent, due to their inhibitory activity and the resistance indices. Furthermore, the effect of guava leaf essential oil against toxoplasmosis caused by the growth of Toxoplasma gondii were reported [ 29 ]. Additionally, guava leaves were proposed for the treatment of diarrhea caused by enteric pathogens, since it showed significant inhibitory activity against Vibrio cholerae and V. parahemolyticus , Aeromonas hydrophila , Escherichia coli , Shigella spp. and Salmonella spp. [ 30 , 31 , 32 ]. It is suppose that the same plant origin and similar extraction procedure makes that these works show comparable inhibition zones for the bacteria tested [ 30 , 31 ], in contrast to the leaves of India and Bangladesh, where MIC values did not show any concordance [ 31 , 32 ] ( Table 1 ). In addition, a reduction was described for S. flexneri and V. cholera invasion and for their adherence to the human laryngeal epithelial cells, and for the production of E. coli heat labile toxin and cholera toxin, as well as their binding to ganglioside monosialic acid enzyme [ 33 ]. Moreover, other studies also demonstrated the antimicrobial effect of some bacteria that cause gastrointestinal disorders by different methods [ 34 , 35 ]. In contrast to previous results [ 20 , 31 ], no inhibition of the hydrodistillation and n-hexane extract was found against E. coli Salmonella spp. [ 31 ] ( Table 1 ).

Furthermore, guava leaf tea helped control of the growth of influenza viruses, including oseltamivir-resistant strains, via the prevention of viral entry into host cells, probably due to the presence of flavonols [ 36 ].

2.1.2. Neoplasms

All the results published regarding anti-cancer properties have been summarized in Table 2 .

In vitro studies against neoplasm.

β-Caryophyllene oxide (CPO); c-jun NH2-terminal kinases (JNK); dichloromethane (DCM); inhibitory concentration (IC 50 ); mammalian target of rapamycin (mTOR); mitogen-activated protein kinases (MAPKs); phosphatidylinositol 3-kinase (PI3K); prostaglandin endoperoxide H synthase (PGHS); protein kinase B (AKT); ribosomal protein S6 kinase beta-1 (S6K1); signal-related kinases (ERKs); tetrazolium (MTT); total flavonoid content (TFC); total phenolic content (TPC); ↑ increases the affect; ↓ decreases the effect.

Kawakami et al. [ 37 ] evaluated the anti-proliferative activity of guava leaf extract in human-colon adenocarcinoma cell line (COLO320DMA). These authors found that the extract depressed the proliferation rate due to the presence of quercetin and quercetin glycosides. Moreover, different extracts were tested on three cancer cell lines (cervical cancer (HeLa), breast cancer (MDA-MB-231), and osteosarcoma (MG-63)). The extracts showed no anti-proliferative activity towards HeLa cells, although they displayed activity against MDA-MB-231 and MG-63, the ether extract being the most effective, followed by methanol and water extracts. However, ether and methanol extracts presented a cytotoxic effect on non-malignant cell Madine Darby canine kidney (MDCK) [ 38 ]. In contrast, an ethanol extract from the stem and leaves reported significant anti-tumor activity on HeLa and colorectal carcinoma (RKO-AS45-1), whereas its effect was less significant for a lung fibroblast cell line (Wi-26VA4) [ 39 ]. This difference could be due to the origin of the leaves, compounds in the steam, or even to the extraction method selected. In this context, an organic guava leaf extract provided molecular evidence of cytotoxic or anti-tumor activity in human breast carcinoma benign cells (MCF-7) and also in murine fibrosarcoma (L929sA) [ 40 ]. A fact worthy to comment is that the difference noticed in the cytotoxic effect on MDA-MB-231 cell line might be because the extraction differs [ 38 , 40 ]. Furthermore, the aqueous extract of budding guava leaves displayed an anti-tumor effect against human prostate epithelial (PZ-HPV-7) and carcinoma (DU-145) cells in view of the cell-killing-rate coefficients, as well as anti-angiogenesis and anti-migration activities, respectively [ 41 , 42 ].

Regarding the bioactivity of terpenes from guava, an enriched mixture of guajadial, psidial A, and psiguadial A and B proved anti-proliferative effect for nine human cancer lines: leukemia (K-562), breast (MCF-7), resistant ovarian cancer (NCI/ADR-RES), lung (NCI-H460), melanoma (UACC-62), prostate (PC-3), colon (HT-29), ovarian (OVCAR-3), and kidney (786-0) [ 43 ]. The apoptotic effect of β-caryophyllene oxide (CPO) on MCF-7 and PC-3 cell lines was also demonstrated because of its ability to interfere with multiple signaling cascades involved in tumor genesis [ 44 ]. Moreover, the essential oil from guava leaves exerted an anti-proliferative effect on human-mouth epidermal carcinoma (KB) and murine leukemia (P388) cell lines [ 45 ], while a hexane fraction of the leaves showed a cytotoxic effect against leukemia (Kasumi-1) cancer-cell line at higher half maximal inhibitory concentration (IC 50 ), probably due to a less concentration of the bioactive compounds of the leaves [ 46 ]. Finally, cytotoxic and apoptotic effect in PC-3 cells and apoptotic effect in LNCaP cells was reported. The lack of cytotoxic effect in LNCaP might be because the cell growth is androgen-dependent, while in PC-3 is androgen-independent. [ 47 ]. Comparing these data with those reported by Park et al. [ 44 ], high concentration is needed for causing cell death, and a weak effect is found on early apoptotic cell. The main difference between these works is the composition of the extract, so it could be concluded that an antagonist effect is produced amongst the isolated compounds by Ryu et al. [ 47 ].

2.1.3. Diseases of the Blood and Immune System

A fermented guava leaf extract was tested in mouse macrophage (RAW 264.7) cells. The results confirmed its potential to decrease the expression of lipopolysaccharide-inducible nitric oxide synthase and cyclooxygenase-2 proteins level, two pro-inflammatory mediators, through the down-regulation of nuclear factor-κB transcriptional activity (NF-κB) [ 48 ]. This biological activity was also reported in other works [ 40 , 49 , 50 ]. Briefly, Jang et al. [ 49 ] evaluating the prostaglandin E 2 production found that the inhibitory effect was highly correlated to the total phenolic content. Kaileh et al. [ 40 ] suggested that the suppression of the nuclear factor-κB could be at the transcriptional level because of the lack of binding between nuclear factor-κB and DNA in murine fibrosarcoma (L929sA) and two breast-cancer cell lines (MDA-MB231 and MCF7). At the same time, Jang et al. [ 50 ] found that the lipopolysaccharide-induced production of nitric oxide and prostaglandin E 2 was due to the ability of guava leaf extract to suppress phosphorylation in protein expression. Moreover, Sen et al. [ 51 ] verified the inhibition of nuclear factor-κB activation in Labeo rohita head-kidney macrophages by the flavonoid fraction of guava leaf extract and Jang et al. [ 52 ] improved the inhibition of lipopolysaccharide-induced prostaglandin E 2 and nitric oxide production by optimizing of the extraction conditions. Furthermore, methanol and ethanol leaf extracts also showed the inhibition of hypotonicity-induced lysis of erythrocyte membrane [ 53 ]. Meanwhile, Laily et al. [ 54 ] suggested the use of guava leaves as immune-stimulant agent because they modulated the lymphocyte proliferation response.

The results for this activity, confirm the potential of guava leaves as an anti-inflammatory treatment and as immune-system stimulatory agent. As is shown in Table 3 , a general trend is reported in every work, although the differences noticed in the data are probably due to the different extraction method and to the doses assayed, or even to the harvesting time of the leaves. However, the mechanism should be further studied since two different pathways are suggested for the down-regulation of NF-κB.

In vitro assays against diseases of the blood and immune system.

Cyclooxygenase-2 (COX-2); dichloromethane (DCM); gallic acid equivalent (GAE); human red blood cell (HRBC); inducible nitric oxide synthase (iNOS); inhibitor of kappa B (I-κBα); interleukin-1β (I-1β); lipopolysaccharide (LPS); mitogen-activated protein kinases (MAPKs); nitric oxide (NO); prostaglandin E 2 (PEG 2 ); reverse transcription-polymerase chain reaction RT-PCR; tetrazolium (MTT); total phenolic content (TPC); transcriptional nuclear factor-κB (NF-κB); Tumor necrosis factor alpha (TNF-α); ↑ increases the affect; ↓ decreases the effect.

2.1.4. Endocrine and Metabolic Diseases

Several works have focused on elucidating the anti-diabetic compounds present in guava leaves ( Table 4 ). Although the origin of the leaves remains different, the presence of these compounds has demonstrated the hypoglycemic effect of the leaves via different assays. However, the main mode of action seems to be due to an inhibition of the enzymes related to this activity.

Compounds in guava leaves with anti-diabetic properties in in vitro assays.

Advanced glycation end products (AGEs); bovine serum albumin (BSA), dipeptidyl peptidase (DP); effective concentration (EC 50 ); glucose transporter 2 and 5 (GLUT-2; GLUT-5); human serum albumin (HSA); inhibitory concentration (IC 50 ); insulin receptor (IR); insulin receptor substrate (p-IRS (Tyr)); p85 regulatory subunit of phospho-inositide 3 kinase (PI3K (p85)); phosphorylation of the insulin receptor (p-IR (Tyr)); protein kinase B (p-Akt (Ser)); tumor necrosis factor (TNF); ↓ decreases the effect.

The anti-glycative potential of the guava leaves was evaluated, with the conclusion that the extract inhibited, in vitro, the formation of advanced glycation end-products formation [ 55 ]. Moreover, the aqueous guava leaf extract, in an albumin/glucose model system, also exerted the same effect and indeed inhibited Amadori products. Gallic acid, catechin and quercetin exhibited over 80% inhibitory effects whereas ferulic acid showed no activity [ 56 ]. In another study, seven pure flavonoid compounds (quercetin, kaempferol, guaijaverin, avicularin, myricetin, hyperin, and apigenin) showed strong inhibitory activities against sucrase, maltase, and α-amylase, and a clear synergistic effect against α-glucosidase [ 57 ]. Moreover, Deguchi and Miyazaki [ 58 ] suggested that the component that inhibited the in vitro activities of α-glucosidase enzymes in guava extract was a polymerized polyphenol. In addition, polysaccharides from guava leaves also exhibited α-glucosidase inhibition [ 59 ].

Eidenberger et al. [ 60 ] demonstrated the dose-dependent inhibition of guava leaf ethanol extracts on dipeptidyl-peptidase-IV due to the individual flavonol-glycosides: peltatoside, hyperoside, methylquercetin hexoside, isoquercitrin, quercetin/morin pentoside, guaijaverin, and quercetin/morin pentoside. Additionally, the individual flavonol-glycosides found in the guava extract reported no significant differences compared with the uptake of the whole guava extract into epithelial cells (CaCo-2) [ 60 ]. In the same cell line, the inhibition of fructose uptake was also tested by Lee et al. [ 61 ], who confirmed that catechin and quercetin contributed to the inhibition of glucose transporters. In addition, the enhancement of aqueous guava leaf extract was investigated with regard to glucose uptake in rat clone 9 hepatocytes. Moreover, quercetin was proposed as the active compound responsible for promoting glucose uptake in liver cells and contributing to the alleviation of hypoglycemia in diabetes [ 62 ]. Furthermore, Basha and Kumari [ 63 ] also estimated the glucose uptake of different extracts. The methanol extract of guava leaves was found to be the most efficient in lowering glucose levels. Basha et al. [ 64 ] demonstrated the ability of guavanoic-acid-mediated gold nanoparticles to inhibit the protein tyrosine phosphatase 1B activity.

Indeed, a guava leaf ethanol extract was tested in pre-adipocyte cell line (3T3-L1), which showed its ability to inhibit adipocyte differentiation via down-regulation of adipogenic transcription factors and markers, and hence may prevent obesity in vivo [ 65 ]. To evaluate the potential of the leaves on glucose uptake and glycogen synthesis, an aqueous extract was used in insulin-resistant mouse (FL83B) cells. The results confirmed the improved expression and phosphorylation of insulin signaling-related proteins, promoting glycogen synthesis and glycolysis pathways. In fact, this work provides new insights into the mechanisms through which the guava extract improves insulin resistance in the hepatocytes [ 66 ]. In the same cell line, vescalagin was postulated as the active component that may alleviate the insulin resistance in mouse hepatocytes [ 67 ].

In this sense, the latest study made in L6 myoblasts and myotubes cells confirmed that the glucose uptake recruitment followed a wortmannin-dependent pathway. In addition, guava leaves also inhibited aldose reductase activity, up-regulated gene- and protein-level expression of several insulin receptors and also improved cellular-level glucose uptake [ 68 ].

2.1.5. Diseases of the Circulatory System

Cardiovascular disorders have been related to the endothelial cell damage that causes atherosclerosis. In this sense, extracts from budding guava leaves demonstrated a protective, in vitro, effect in bovine aortal endothelial cells, delaying low-density lipoprotein oxidation and preventing oxidized low-density lipoprotein cytotoxicity [ 69 ]. A similar effect was also noted in human umbilical-vein endothelial cell due to the ability of saving cell-viability reduction, suppressing reactive oxygen species production and nitric oxide release, as well as inhibiting the expression of NF-κB [ 70 ]. Moreover, budding guava leaves also showed their ability as an anticoagulant in plasma, since they reduced thrombin clotting time and inhibited the activity of antithrombin III. Thus, they could help to reduce the development of cardiovascular complications [ 71 ].

In addition, flavonoids and phenolic acids in the leaves could contribute to the prevention and amelioration of gout and hypertension, since, in rat-tissues homogenates, they inhibit the activity of two enzymes related to the development of both diseases (xanthine oxidase and angiotensin 1-converting enzymes) [ 72 ].

2.1.6. Diseases of the Digestive System

Guaijaverin, isolated from guava leaves, displayed high inhibitory activity against Streptococcus mutans . In fact, guaijaverin exhibited its ability as an anti-plaque agent, becoming an alternative for oral care [ 73 ]. Furthermore, guava leaves showed greater bactericidal effect on early ( Streptococcus sanguinis ) and late ( S. mutans ) colonizers compared to Mangifera indica L. and Mentha piperita L. leaves, whereas, when they are compared with the plant extract mixture, the effect is slightly lower. By contrast, guava leaves showed similar and higher anti-adherence effect than the plant mixture [ 74 ]. In another study, the whole extract was tested on the cell-surface hydrophobicity of selected early settlers and primary colonizers of dental plaque, showing its ability to alter and disturb the surface characteristics of the agents, making them less adherent [ 75 , 76 , 77 ], and also delayed in the generation of dental biofilm by targeting growth, adherence, and co-aggregation [ 78 ]. This property could be due to the presence of flavonoids and tannins detected in P. guajava [ 79 ]. Shekar et al. [ 80 ] also confirmed the use of the leaves as anti-plaque agents against Streptococcus mutans , S. sanguinis , and S. salivarius . Kwamin et al. [ 81 ] discovered the effectiveness of guava leaf extract in the leukotoxin neutralization of Aggregatibacter actinomycetemcomitans , leading it to be considered as a possible agent for the treatment of aggressive forms of periodontitis. In addition, extracts rich in guava flavonoids have demonstrated their potential for preventing dental caries due to the growth inhibition of the oral flora [ 82 ]. Moreover, its soothing of toothache has been verified based on the analgesic, anti-inflammatory, and anti-microbial activity properties [ 83 ] and it has been reviewed positively as an adjutant for treating periodontal disease [ 84 ].

Concerning the liver disorders, the cytotoxic and hepato-protective effects of guava leaves were reported. Studies carried out in clone 9 cells treated with different extracts of the leaves showed that only ethanol and acetone extracts tend to have cytotoxicity effect at high concentrations. Moreover, the ethanol extract showed hepato-protective activity, although the hot-water extract reported greater effect and lower cytotoxicity [ 85 ].

Table 5 compiles the methodology followed and the results reported in the present works. It is important to keep in mind that the origin, the selection of the extraction method or solvent, and the concentration of the extract tested generally provide different data. For example, comparing data for inhibition zones, best results are noticed at long maceration time in acetone, which seems to be a better extracting solvent than ethanol [ 77 , 78 , 80 , 82 ]. Hydrophobicity depends on the origin of the leaves, the extraction method, and the concentration of the extract tested, and it also depends on the lipophilic (index > 70%) or hydrophilic nature of the strain [ 73 , 75 , 79 ]. Finally, minimum inhibitory concentration relies on all factors.

In vitro assays against diseases related to the digestive system.

Alanin aminotransferase (ALT); colony forming unit (CFU); human red blood cell (HRBC); microbial adhesion to hydrocarbon test (MATH); minimum bactericidal concentration (MBC); minimum fungicidal concentration (MFC); minimum inhibitory concentration (MIC); nordini’s artificial mouth (NAM); Tetrazolium (WST-1); ↑ increases the affect; ↓ decreases the effect.

2.1.7. Diseases of the Skin and Subcutaneous Tissue

Qa’dan et al. [ 86 ] described the antimicrobial effect of a leaf extract against the main developer of acne lesions, Propionibacterium acnes , and other organisms isolated from acne lesions. The antimicrobial activity was also displayed against pathogenic bacteria associated with wound, skin, and soft-tissue infections [ 87 ]. Furthermore, antifungal properties have also been studied by Padrón-Márquez et al. [ 88 ]. The acetone and methanol extracts displayed relevant activity against dermatophytic fungi, and thus could be considered as new agents against skin disease. Furthermore, phenols from the leaves were tested on human-skin fibroblast cells and showed antifungal properties [ 89 ].

In addition, the tyrosinase inhibitory activities of 4 different parts (branch, fruit, leaf, and seed) of guava, extracted with acetone, ethanol, methanol, and water were tested by You et al. [ 90 ] who reported that the ethanol extract from the leaves reached the highest activity. Therefore, the leaves might be appropriate for both boosting the whitening of skin and inhibiting browning. In addition, in a human keratinocyte cell line, an ethyl acetate extract showed a positive effect on atopic dermatitis via the inhibition of cytokine-induced Th2 chemokine expression [ 91 ].

Lee et al. [ 92 ] carried out the first electrophysiological study based on ultraviolet (UV)-induced melanogenesis with guava leaves. The authors suggested the use of guava leaves for both direct and indirect prevention of skin melanogenesis caused by UV radiation. In fact they demonstrate that methanolic guava leaves extract inhibits tyrosinase, that is the key enzyme in melanin synthesis, and ORAI1 channel that has shown to be associated with UV-induced melanogenesis.

2.1.8. Other Activities Related to Several Diseases

An aqueous guava extract showed its ability to decrease the radiolabeling of blood constituent due to an antioxidant action and/or because it alters the membrane structures involved in ion transport into cells [ 93 ]. Guava leaves also have been demonstrated to possess anti-allergic effects in rat mast (RBL-2H3) cell line by the inhibition of degranulation and cytokine production, as well as blocking high-affinity immunoglobulin E-receptor signaling [ 94 ].

2.2. In Vivo Studies

2.2.1. infectious and parasitic diseases.

After checking the effect of guava leaf extract, in vitro, against Aeromonas hydrophila , in vivo experiments were carried out in tilapia ( Oreochromis niloticus ), indicating the potential use of P. guajava as environmentally friendly antibiotic [ 95 ]. The leaves also had anti-viral and anti-bacterial activity towards shrimp ( Penaeus monodon ) pathogens such as yellow-head virus, white spot syndrome virus, and Vibrio harvey . In addition, guava leaf extract improved the activities of prophenoloxidase and nitric oxide synthase in serum, and of superoxide dismutase, acid phosphatase, alkaline phosphatase, and lysozyme in serum and hepatopancreas [ 96 ].

Furthermore, guava leaves have been suggested for managing sleeping sickness, since they exhibited trypanocidal effect in albino rats [ 97 ]; the extract ameliorate the tissue-lipid peroxidation associated to trypanosomosis, as well as raising the level of the glutathione concentration [ 98 ]. The leaves also showed anti-malarial effect in BALB/c mice infected with Plasmodium berghei via parasitemia suppression [ 99 ]. Moreover, guava leaves are also recommended for treating infectious diarrhea since they prevented intestinal colonization of Citrobacter rodentium in Swiss albino mice [ 100 ]. In chicks, guava leaf extract enabled the control of diarrhea produced by E. coli and reduced the severity of its symptomatology [ 101 ]. In mice, the improvement of cholera symptoms caused by V. cholerae , a human pathogen, was also confirmed by Shittu et al. [ 102 ].

In addition, anti-helminthic properties towards gastro-intestinal nematodes have been found, as a result of the presence of condensed tannins in the guava plant, which raised the levels of hemoglobin, packed cell volume, total protein, globulin, glucose, and calcium, and lowered the levels of blood urea [ 103 ].

All the results published regarding in vivo anti-bacterial properties have been summarized in Table 6 .

In vivo anti-bacterial effect.

Acid phosphatase (ACP); alkaline phosphatase (AKP); colony forming unit (CFU); globulin (GLO); glutathione (GSH); hemoglobin (Hb); lysozyme (LSZ); malondialdehyde (MDA); median lethal dose (LD 50 ); nitric oxide synthase (NOS); packed cell volume (PCV); prophenoloxidase (PO); superoxide dismutase (SOD); ↑ increases the affect; ↓ decreases the effect.

2.2.2. Neoplasms

Only one study is available on the anti-tumor effect that could be related to the phenolic composition of guava leaves. An ethanol extract of the leaves was administrated to B6 mice after inoculation of melanoma cells. The results suggested that the extract had a vaccine effect, but not a therapeutic effect, against tumors through by depressing T regulatory cells [ 104 ].

Moreover, the meroterpene-enriched fraction of guava leaves, containing guajadial, psidial A, and psiguadial A and B, was evaluated in vivo in a solid Ehrlich murine breast-adenocarcinoma model. The results suggested that these compounds may act as phytoestrogens, presenting tissue-specific antagonistic and agonistic activity on estrogen receptors [ 43 ]. These data partially confirmed the results in vitro obtained by Ryu et al. [ 47 ].

2.2.3. Diseases of the Blood and Immune System

Among blood diseases, anemia indicates a failure in the immune system. In this sense, guava extract presented an anti-anemic effect in trypanosomosis-infected Wistar rats, improving the values of hemoglobin, packed cell volume, red-blood cell counts, mean corpuscular volume, and mean concentration hemoglobin count while decreasing white-blood cell and neutrophil levels [ 105 ]. Moreover, the same trend in the hematological analyses was also recorded in mice. After the administration of guava leaf extract, no alterations on the erythron were detected [ 106 ]. Nevertheless, results differ because subjects under study are different, also the duration of the treatment, the extraction method and the dose assayed ( Table 7 ).

In vivo studies against diseases of the blood and immune system.

Hemoglobin (Hb); interleukin-6 (IL-6); lipopolysaccharide (LPS); mean concentration hemoglobin count (MCHC); mean corpuscular volume (MCV); packed cell volume (PCV); paw withdrawal latency (PWL); red-blood cell counts (RBCC); tumor necrosis factor alpha (TNF-α); white-blood cell (WBC); ↑ increases the affect; ↓ decreases the effect.

The anti-inflammatory response of the leaves was dose-dependent in induced hyperalgesia in Sprague-Dawley rats, decreasing in paw-withdrawal latency, and significantly improving the survival rate of mice with lethal endotoxemia [ 50 ]. Moreover, the anti-inflammatory activity of aqueous and acetone–water extracts of the leaves was also confirmed in Swiss mice by reducing the amount of leukocyte migration. The acetone–water extract also exhibited peripheral analgesic activity, probably by blocking the effect or the release of endogenous substances that excite pain-nerve endings [ 19 ]. The analgesic effect in albino rats was also reported. The ethanol extract reduced the writhing response [ 107 ], and a jumping response was found after the administration of a distilled extract (combination of methanol and aqueous extracts) [ 108 ]. In this case, the writhing response for both Swiss mice and Wistar rats seems to be comparable, although the dose assayed is completely different ( Table 7 ).

2.2.4. Endocrine and Metabolic Diseases

Guava leaves have shown their potential against one of the diseases with the highest incidence level worldwide, diabetes mellitus, and also towards biochemical changes caused by the disease. In spite of being leaves from different countries, treatments in different subjects or even different data, the same trend is followed in these works ( Table 8 ).

Endocrine and metabolic in vivo assays with guava leaves.

acid phosphatase (ACP); alanine aminotransferase (ALT); alkaline phosphatase (ALP); aspartate aminotransferase (AST); catalase (CAT); glutathione peroxidase (GPx); glutathione reductase (GRd); high-density lipoprotein (HDL) cholesterol; low-density lipoprotein (LDL) cholesterol; protein tyrosine phosphatase 1B (PTP1B); superoxide dismutase enzyme (SOD); total cholesterol (TC); triglycerides (TG); very low-density lipoprotein (VLDL) cholesterol; ↑ increases the affect; ↓ decreases the effect.

The effect of aqueous guava leaf extract was investigated in rabbits, fed a high-cholesterol diet. Treatment with guava leaves reduced the plasma-cholesterol level, caused a remarkable spike in high-density lipoprotein, a dip in low-density lipoprotein levels, and significantly reduced the associated hyperglycemia. In addition, the extract showed hypolipidemic and hypoglycemic potentials in hypercholesterolemic rabbits [ 109 ]. Furthermore, guava leaves reduced oxidative stress induced by hypercholesterolemia in rats [ 110 ].

In addition, the anti-diabetic effect was also evaluated in Lepr db / Lepr db mice and significant blood-glucose-lowering effects were observed. In addition, histological analysis revealed a significant reduction in the number of lipid droplets, which, furthermore, at least in part, could be mediated via the inhibition of protein tyrosine phosphatase 1B [ 111 ].

In streptozotocin-induced diabetic rats, the administration of oral doses of aqueous and ethanol extracts from guava leaves could alter the Ca:Mg ratio [ 112 ]; however, in low-dose streptozotocin and nicotinamide-induced Sprague-Dawley diabetic rats, long-term administration of guava leaf extracts raised the plasma-insulin level, the glucose utilization, and the activity of hepatic enzymes [ 113 ]. Moreover, the leaves also lowered blood glucose levels and decreased protein glycation [ 55 ].

In agreement with the above, a lower blood-glucose level was also reported in alloxan-induced diabetic rats. Additionally, no side effects were observed in certain liver enzymes (alkaline phosphatase and aspartate aminotransferase) whereas alanine aminotransferase activity declined [ 114 ]. In alloxan-induced diabetic rats, a decrease was also found in blood glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and a significant increase in high-density lipoprotein cholesterol after 21 days of treatment with guava leaf ethanolic extract [ 115 ].

Among the works that evaluated only biochemical parameters, guava leaf extract promoted changes due to an alteration on the activity of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and acid phosphatase in the kidney, liver, and serum [ 106 , 116 ]. In addition, Adeyemi and Akanji [ 117 ] evaluated the effect of daily administration of guava leaves, demonstrating the alteration of the serum homeostasis and the pathological variations in rat tissues.

2.2.5. Diseases of the Circulatory System

Ademiluyi et al. [ 118 ] assessed the lipid peroxidation in rats after checking the antihypertensive effect, in vitro, of red and white guava leaves. The work concluded that the activity may be related to rosmarinic acid, eugenol, carvacrol, catechin, and caffeic acid since they were the major constituents of their extracts. In addition, this activity was supported by the biphasic and contractile effect on rat vascular smooth muscles [ 119 , 120 ].

In addition, atherosclerosis development was reduced in apoE-knockout mice by guava leaf extracts. In fact, the effect was connected to the presence of ethyl gallate and quercetin [ 121 , 122 ]. In streptozotocin-induced diabetic rats, vascular reactivity to vasoconstrictor agents was reduced, as was vessel atherosclerosis [ 112 ]. Furthermore, Soman et al. [ 123 ] found that an ethyl acetate fraction of guava leaves reduced cardiac hypertrophy in streptozotocin-induced diabetic rats due to an anti-glycative effect.

2.2.6. Diseases of the Digestive System

In the digestive system, formed by the gastrointestinal tract plus the group organs necessary for the digestion, guava leaves have demonstrated activity towards different parts.

On the one hand, the leaves have shown the ability to protect the stomach against ulceration by inhibiting gastric lesions, reducing gastric secretory volume, and acid secretion, and raising the gastric pH [ 124 , 125 , 126 ]. This anti-ulcer activity, resulting from the protection of the mucosa, was related to the flavonoids in the leaves [ 127 ]. Despite of the subject employed for the assay, similar data are reported in these works ( Table 9 ). The anti-diarrheal activity of guava leaf aqueous extract was evaluated on experimentally induced diarrhea in rodents. The extract performed in the same way as the control drugs, offering protection, inhibiting intestinal transit, and delaying gastric emptying [ 128 ]. Another study attributed this activity to a dual action between the antimicrobial effect and the reduction in gastrointestinal motility ability of the extract [ 129 ]. In rabbits, the anti-spasmodic effects were connected to a calcium channel blocking activity, which explains the inhibitory effect on gut motility. The anti-diarrheal protection was also tested in mice [ 130 ]. As is shown in Table 9 , the anti-diarrheal activity is dose-dependent, although the protection varied depending on the subjects.

In vivo assays for digestive system related diseases.

Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); aspirin (ASP); aspartate aminotransferase(AST); catalase (CAT); carbon tetrachloride (CCl 4 ); ethanol (EtOH); gamma glutamyl transferase (GGT); gastric volume (GV); globulin (GLO); glutathione (GSH); glutathione peroxidase (GPx); glutathione S-transferase (GST); lactate dehydrogenase (LDH); lipid peroxidation (LPO); mean number lesions (MNL); paracetamol (PCM); pyloric ligation (PL), Serum glutamic oxaloacetic transaminase (SGOT); Serum glutamic pyruvic transaminase (SGPT), superoxide dismutase (SOD); thioacetamide (TAA); ulcer index (ui); ↑ increases the affect; ↓ decreases the effect.

On the other hand, guava leaves exhibited hepato-protective effect due to the reduction of serum parameters of hepatic enzymes markers and histopathological alterations in the acute liver damage induced in rats [ 131 , 132 , 133 , 134 , 135 ]. Here, a dose-dependent effect is also found. However, decoction of the leaves seems to be the best option for the extraction of the compounds that exhibited this activity ( Table 9 ).

2.2.7. Diseases of the Skin and Subcutaneous Tissue

Guava leaves have been suggested as a therapeutic agent to control pruritus in atopic dermatitis. The improvement of the skin lesions was due to a reduction in serum immunoglobulin E level and in the eczematous symptoms [ 136 ]. Moreover, the epithelium was repaired with connective tissue and absence or moderate presence of inflammatory cells by the leaves. As a result, the leaves exhibited wound healing properties [ 137 ]. Furthermore, guava leaf extract was tested on rat skin, and exhibited inhibitory activity towards an active cutaneous anaphylaxis reaction [ 138 ].

2.2.8. Other Activities Related to Several Diseases

Triterpenoids from guava leaves were suggested as a potential therapeutic approach for treating diabetic peripheral neuropathy, as they enhanced physical functions and offered neuronal protection towards the suppression of the expression of pro-inflammatory cytokines [ 139 ]. In addition, the leaves can act as radio modulators for cancer patients because by preventing DNA damage and apoptosis. [ 140 ], as well as protective agents by restoring the normal values of sperm viability, sperm count, sperm motility, and sperm-head abnormality caused by caffeine-induced spermatotoxicity [ 141 ].

Moreover, the consumption of guava leaf tea was evaluated, in vivo, in the inhibition of cytochrome P450 (CYP) 3A-mediated drug metabolism by the interaction between guava tea and several drugs [ 11 , 142 ]. Matsuda et al. [ 11 ] investigated the consequence of the ingestion of guava tea for two weeks in rats, and the effect with an anxiolytic drug. The short-term consumption of the tea had little effect on the assays performed. This weak influence was due to the absence of interaction between the tea and midazolam in the metabolism studied. In addition, two in vivo studies were made in rats, to evaluate the interaction of guava leaf tea with an anti-coagulant drug (warfarin) [ 142 ]. Kaneko et al. [ 141 ] suggested that because the tea contained compounds that block the affinity between the enzyme and phenolic compounds of the tea, long-term administration showed a low probability of causing drug-metabolizing enzymes. Moreover, short-term administration revealed that the tea did not interfere with coagulation, meaning that the tea consumption did not alter the pharmacological effect and displayed no side effects.

2.3. Clinical Trials

To test the effect of guava leaf extract, several randomized clinical trials have been conducted during the last two decades, although only two studies are available in the last decade. One of the studies consisted of evaluating the effect of guava leaf extract pills on primary dysmenorrhea disorder. For this, 197 women were divided into four groups, and each received a different dosage: 3 and 6 mg extract/day, 300 mg placebo/day and 1200 mg ibuprofen/day. The administration took place over five days during three consecutive cycles. The results demonstrated that 6 mg extract/day alleviated menstrual pain and could replace the use of medicaments like ibuprofen. In fact, guava leaves could be used as a broad-spectrum phyto-drug and not only as an anti-spasmodic agent [ 143 ]. Furthermore, Deguchi and Miyazaki [ 58 ] reviewed several works regarding the effect of the intake of a commercial guava leaf tea (Bansoureicha ® , Yakult Honsha, Tokyo, Japan) on different pathologies of diabetes mellitus illness such as the influence on postprandial blood glucose, on insulin resistance and on hypertriglyceridemia and hypercholesterolemia. The authors concluded that the ingestion of guava leaf tea can ameliorate the symptoms of diabetes mellitus and that it could be used as an alimentotherapy.

3. Other Applications

Further applications found with guava leaves are listed below: firstly, to prepare gelatin beads with marine-fish gelatin for various applications such as medicine, and the food and pharmaceutical industries [ 144 ]. Secondly, Giri et al. [ 145 ] suggested guava leaves as supplementary feed for the fish species Labeo rohita , due to the immune-stimulatory effect. The same conclusion was reached by Fawole et al. [ 146 ] in L. rohita . Thirdly, Gobi et al. [ 147 ] reported that guava leaf powder, mixed with a commercial diet, strengthened the immunological response of Oreochromis mossambicus , and recommended the leaves as feed complement in aquaculture.

4. Conclusions

Traditional claims generally require experimental research to establish their effectiveness. In this regard, ethnomedicine applications of Psidium guajava L. leaves have been verified by several researches over the last decade against many disorders, demonstrating its potential in the treatment of the most common worldwide diseases. In addition, the effects of the leaves have been related to individual compounds such as quercetin, catechin, vescalagin, gallic acid, peltatoside, hyperoside, isoquercitrin, and guaijaverin.

Future prospects should be aimed at investigating the biodiversity of guava and/or the purification of the different compounds present in guava leaves in order to obtain functional ingredients for further uses as alternative agents in natural therapeutic approaches.


The author Vito Verardo thanks the Spanish Ministry of Economy and Competitiveness (MINECO) for “Ramony Cajal” post-doctoral contract.

Author Contributions

Elixabet Díaz-de-Cerio contributed to the literature review and manuscript redaction; Vito Verardo and Ana María Gómez-Caravaca contributed to the conception of the idea and framework writing; and Alberto Fernández-Gutiérrez and Antonio Segura-Carretero supervised the progress of work.

Conflicts of Interest

The authors declare no conflict of interest.

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Potency of Guava Leaf Extract (Psidium guajava L.) as a Cosmetic Formulation: A Narrative Literature Review

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Guava ( P. guajava L., Myrtaceae ) is one of the fruit plants that is widespread in the tropics and subtropics, including South America, Africa and Asia. Traditionally this plant is often used as food, traditional medicine, dyes and others.  Based on the literature review of the guava leaf plant ( P. guajava L.) has many bioactive compounds that play a role in maintaining the health of the body's skin. Therefore, this plant has the potential to be the basic ingredient for the formulation of cosmetic preparations. This riview literature aims to find out thetension of guava leaves ( P. guajava L . ) as a basic ingredient in the manufacture of natural ingredients cosmetics. The results of this literature review show that guava leaves ( P. guajava L.)  has many benefits for cosmetics, namely guava leaves ( P. guajava L.)  can be used as an anti-acne cleanser, body scrub, lotion, deodorant, toner, and face cream.

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Guava ( psidium guajava l.) leaves: nutritional composition, phytochemical profile, and health-promoting bioactivities.

review of related literature guava leaves extract

1. Introduction

2. chemical composition, 2.1. proximate composition, 2.1.1. polysaccharides, 2.1.2. proteins, 2.1.3. minerals and vitamins, 2.2. phytochemical profile, 2.2.1. essential oil profile, 2.2.2. phenolic compounds, 3. biological activities of guava leaf extracts, 3.1. anticancer/antitumor activity, 3.2. antidiabetic activity, 3.3. antioxidant activity, 3.4. antidiarrhea activity, 3.5. antimicrobial activity, 3.6. hepatoprotective properties, 3.7. antiobesity and lipid-lowering activity, 4. gls as a functional food ingredient, 5. conclusions and future perspectives, author contributions, conflicts of interest.

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Kumar, M.; Tomar, M.; Amarowicz, R.; Saurabh, V.; Nair, M.S.; Maheshwari, C.; Sasi, M.; Prajapati, U.; Hasan, M.; Singh, S.; Changan, S.; Prajapat, R.K.; Berwal, M.K.; Satankar, V. Guava ( Psidium guajava L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health-Promoting Bioactivities. Foods 2021 , 10 , 752. https://doi.org/10.3390/foods10040752

Kumar M, Tomar M, Amarowicz R, Saurabh V, Nair MS, Maheshwari C, Sasi M, Prajapati U, Hasan M, Singh S, Changan S, Prajapat RK, Berwal MK, Satankar V. Guava ( Psidium guajava L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health-Promoting Bioactivities. Foods . 2021; 10(4):752. https://doi.org/10.3390/foods10040752

Kumar, Manoj, Maharishi Tomar, Ryszard Amarowicz, Vivek Saurabh, M. Sneha Nair, Chirag Maheshwari, Minnu Sasi, Uma Prajapati, Muzaffar Hasan, Surinder Singh, Sushil Changan, Rakesh Kumar Prajapat, Mukesh K. Berwal, and Varsha Satankar. 2021. "Guava ( Psidium guajava L.) Leaves: Nutritional Composition, Phytochemical Profile, and Health-Promoting Bioactivities" Foods 10, no. 4: 752. https://doi.org/10.3390/foods10040752

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Original research article, effects of guava ( psidium guajava l.) leaf extract on the metabolomics of serum and feces in weaned piglets challenged by escherichia coli.

review of related literature guava leaves extract

The effects of dietary supplementation with guava leaf extracts (GE) on intestinal barrier function and serum and fecal metabolome in weaned piglets challenged by enterotoxigenic Escherichia coli (ETEC) were investigated. In total, 50 weaned piglets (Duroc × Yorkshire × Landrace) from 25 pens (two piglets per pen) were randomly divided into five groups: BC (blank control), NC (negative control), S50 (supplemented with 50 mg kg −1 diet GE), S100 (100 mg kg −1 diet GE), and S200 (200 mg kg −1 diet GE), respectively. On day 4, all groups (except BC) were orally challenged with enterotoxigenic ETEC at a dose of 1.0 × 10 9 colony-forming units (CFUs). After treatment for 28 days, intestinal barrier function and parallel serum and fecal metabolomics analysis were carried out. Results suggested that dietary supplementation with GE (50–200 mg kg −1 ) increased protein expression of intestinal tight junction proteins (ZO-1, occludin, claudin-1) ( p < 0.05) and Na + /H + exchanger 3 (NHE3) ( p < 0.05). Moreover, dietary supplementation with GE (50–200 mg kg −1 ) increased the level of tetrahydrofolic acid (THF) and reversed the higher level of nicotinamide-adenine dinucleotide phosphate (NADP) induced by ETEC in serum compared with the NC group ( p < 0.05), and enhanced the antioxidant capacity of piglets. In addition, dietary addition with GE (100 mg kg −1 ) reversed the lower level of L -pipecolic acid induced by ETEC in feces compared with the NC group ( p < 0.05) and decreased the oxidative stress of piglets. Collectively, dietary supplementation with GE exhibited a positive effect on improving intestinal barrier function. It can reprogram energy metabolism through similar or dissimilar metabolic pathways and finally enhance the antioxidant ability of piglets challenged by ETEC.


Weaned piglets infected with enterotoxigenic Escherichia coli (ETEC) may cause post-weaning diarrhea, which leads to growth retardation and damage to the innate and adaptive immune systems of piglets. These risk factors increase the morbidity and mortality of piglets and result in large economic losses in the swine industry worldwide ( 1 , 2 ). Regarding the mechanisms of ETEC infectious diarrhea, it has been demonstrated that ETEC can produce colonization factors (CFs) and enterotoxins that adhere to the intestinal mucosa of piglets, and this action inhibits intestinal immune function, perturbs hydro-electrolytic secretions in the intestine, and results in the occurrence of diarrhea ( 3 ).

As a consequence of this, veterinary antibiotics have been commonly used to treat intestinal infections for improving animal growth and health in several decades. However, concerns about antimicrobial resistance, residue accumulation in animal products, and environmental pollution have led to a limited application of antibiotics as growth promoters ( 4 , 5 ). Due to these factors, searching for alternatives to antibiotic growth promoters, such as pro- and prebiotics, organic acids, enzymes, and plant extracts, have attracted more and more attention ( 5 – 7 ). Among the candidate alternatives to antibiotics, plant extracts appear to be one of the most widely accepted ( 8 , 9 ).

Guava ( Psidium guajava L.) is a tropical fruit and medicinal plant, which is mainly distributed in the tropical and subtropical areas. Guava leaf extract (GE), known as an herbal medicine for the treatment of respiratory and gastrointestinal diseases ( 10 ), is reported to contain phenolics, triterpenoids, and other compounds that have antibacterial, antioxidant, and anti-inflammatory activities ( 11 – 13 ). Pruning usually is used to stimulate growth and influence fruiting in guava ( 14 ); thus, residual guava leaves from pruned processing are promising sources of natural feed additives, which may be utilized as a potential alternative for in-feed antibiotics.

Current studies have demonstrated that GE possesses antidiarrheal activity in various diarrhea models ( 15 , 16 ). Our previous study also indicated that GE could attenuate diarrhea and improve intestinal anti-inflammatory ability in piglets challenged by ETEC ( 17 ). However, there are few reports about the relationship between the antidiarrheal effect of GE and related metabolic regulation. In the post-genomic era, metabolomics is an emerging strategy of research in the field of biological sciences, which provides a platform to study the endogenous metabolite changes in response to a biological system with genetic or environmental changes ( 18 ). In fact, metabolomics may shed light on the complex interaction mechanism between the intestinal diarrhea disease and metabolic phenotype and can regulate them to obtain therapeutic benefits ( 19 ). Therefore, here, we analyzed the intervention of GE on metabolic profiling and related endogenous differential metabolites by metabolomics in weaned piglets. Meanwhile, we also evaluated the effects of GE on tight junction-related proteins in weaned piglets, aiming to provide a potential window through which to explore the crosstalk between GE-mediated metabolic changes and its antidiarrheal processes during the progression of weaned piglets challenged by ETEC.

Materials and Methods

Preparation of guava leaf extract.

Fresh guava leaves were collected from the guava plantations in Qionghai City of Hainan Province, China, during the pruning period in July 2018, and fresh leaves were dried (60°C, 24 h) and powdered. The powder (50 kg) was exhaustively extracted with 95% ethanol three times at room temperature and then filtered. The solvent was evaporated under reduced pressure using a rotary vacuum evaporator to afford GE (3.47 kg), which was stored at 4°C for an animal experiment.

Feeding Trial and Experimental Design

The current feeding trial is from our previous published study ( 17 ). Fifty 21 ± 3 day-old crossbred weaned piglets (Duroc × Yorkshire × Landrace, 7.35 ± 0.18 kg) were selected and transported from the piggery to the barn, where they were randomly allotted to five groups of five replicate pens per group (two piglets per pen). The five groups were as follows: (1) blank control group (BC), piglets were fed diet without supplements and ETEC challenge; (2) negative control group (NC), piglets were fed diet without supplements and challenged by ETEC; (3) S50 group (S50), piglets were fed diet supplemented with 50 mg kg −1 GE and challenged by ETEC; (4) S100 group (S100), piglets were fed diet supplemented with 100 mg kg −1 GE and challenged by ETEC; (5) S200 group (S200), piglets were fed diet supplemented with 200 mg kg −1 GE and challenged by ETEC. The diet was formulated to meet the nutrient recommendations of the National Research Council (2012). The ingredient and nutrient composition of basal diet were presented in Supplementary Table 1 .

Feed and water were available ad libitum during the 28-day experimental period. All piglets were housed in a weaner facility temperature maintained at 25 ± 0.5°C, with 12 h of light and dark. On day 4, all piglets (except BC) were orally challenged with about 1.0 × 10 9 colony-forming units (CFUs) of ETEC according to the method developed by Wu et al. ( 20 ). ETEC was obtained from the China Veterinary Culture Collection Center (Beijing, China). The occurrence of diarrhea during the whole experiment for each group was observed.

Sample Collection

On day 29, one pig was randomly selected from each pen, and the blood samples were collected from the jugular vein, and serum was prepared by centrifugation at 700 × g for 15 min at 4°C and stored at −80°C until metabolomics analysis. After sampling, all piglets were anesthetized by an intraperitoneal injection of 50 mg kg −1 pentobarbital sodium and were killed by exsanguination. Fecal samples were collected directly in 10-ml sterile plastic tubes from the rectum of piglets and stored at −80°C until analysis. The small intestine was removed, and a piece (4-cm length) of the middle jejunum was collected, gently rinsed with 0.1 M phosphate-buffered saline (PBS) at pH 7.2, and then fixed in 10% formaldehyde-phosphate buffer for subsequent immunohistochemical analysis.


Immunohistochemical assay was used to detect the claudin-1, occludin, zonula occludens 1 (ZO-1), and Na + /H + exchanger 3 (NHE3) proteins expression in the jejunal mucosa with densitometric analysis as described previously ( 21 ). Polyclonal primary antibodies against claudin-1, occludin, ZO-1, and NHE3 (1:200 dilution, Proteintech, Wuhan, China) were employed. The average integrated optical density of the positive products was detected by using the Image-Pro Plus software (version 6.0 for Windows) at 200 × magnification.

Serum and Fecal Sample Preparation and Analysis by Ultra-High-Performance Liquid Chromatography Coupled With a Hybrid Quadrupole Time-of-Flight Mass Spectrometry

Serum and fecal samples were extracted prior to analysis by ultra-high-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) in positive ionization mode. Serum samples (100 μl) were prepared via one-step protein precipitation with 400 μl of methanol (TEDIA, Fairfield, USA). The samples were left at −80°C for 6 h after a 2-min vortex. After that, the samples were centrifuged at 20,000 × g for 10 min at 4°C. Then 400 μl of supernatant was transferred into an Eppendorf Tube, and the supernatant was concentrated in a vacuum centrifugal concentrator for 1 h using SPD121P SpeedVac Concentrator (Thermo Fisher, Germany), then reconstituted with 100 μl of acetonitrile (ACN) (MERCK, Darmstadt, Germany) for UHPLC-QTOF-MS analysis as described previously ( 22 ).

For extraction of the fecal samples, fecal samples (100 mg) were prepared via protein precipitation with 500 μl of methanol. Then the samples were vigorously vortexed for 5 min and centrifuged at 20,000 × g at 4°C for 10 min. After the supernatant was collected, the residue again was extracted according to the above extraction procedure and combined with the previous supernatant. At last, 200 μl of supernatant was transferred into the sampling vial for UHPLC-QTOF-MS analysis as described previously ( 23 ).

Briefly, the prepared sample (5 μl) was injected into an XBridge HILIC (2.1 × 100 mm, 3.5 μm) column (Waters, USA) at 30°C in an LC-30AD series UHPLC system (Nexera TM , Shimadzu, Japan), coupled with QTOF mass spectrometer (TripleTOF5600, Sciex, USA, Concord, ON) equipped with Turbo V ESI (electrospray ionization) sources. The mass spectrum was scanned and collected ( m/z 70–1,000) in positive mode at a flow rate of 0.25 ml min −1 . The chromatographic gradient condition for samples analysis was 80–20% B over 0–24 min, 20–80% B over 24–24.5 min, and the composition was held at 80% B for 8.5 min, where A = 50% ACN, and 50% water contains 0.1% formic acid (SIGMA, Deisenhofen, Germany), and B = 95% ACN, and 5% water contains 0.1% formic acid.

Drying gas temperature and the ion spray voltage were set at 550°C and 5,000 V, respectively. Atomization gas pressure, auxiliary heating gas pressure, and curtain gas pressure were set at 45, 45, and 35 psi, respectively. The instrument was mass calibrated by automatic calibration infusing the Sciex Positive Calibration Solution (Sciex, Foster City, CA, USA) after every six-sample injections. One quality control sample and one blank vial were run after each cycle of 10 samples.

Automatic peak extraction, peak matching, peak alignment, and normalization preprocessing on the acquired data were performed using MarkerView software (Sciex, USA). The retention time and m/z tolerances were 0.1 min and 10 ppm, respectively; the response threshold was 100 counts and the isotope peak was removed. After Pareto scaling, principal component analysis (PCA), and partial least-squares discrimination analysis (PLS-DA) models were carried out to visualize the metabolic difference among BC, NC, S50, S100, and S200 groups. The quality of the models was described using R 2 X and R 2 Y. To avoid model overfitting, 999 cross-validations in SIMCA-P 13.0 were performed throughout to determine the optimal number of principal components. R 2 X, R 2 Y, and Q 2 Y values of models were nearly 1.0, indicating that these models retain the ability to explain and predict variations in the X and Y matrix.

Furthermore, the value of fold change (FC) was calculated as the average normalized peak intensity ratio between the two groups. Differences between data sets with FC > 1.10 or and p < 0.05 (Student t -test) were considered statistically significant. The structural identification of differential metabolites was performed by matching the mass spectra with an in-house metabolite library, including accuracy mass, retention time, MS/MS spectra, and online databases Metlin ( http://www.metlin.scripps.edu ) and HMDB ( http://www.hmdb.ca ).

The impact of ETEC and GE on metabolic pathways was evaluated based on the MetaboAnalyst platform, a tool for metabolomics data analysis platform, which is available online ( https://www.metaboanalyst.ca ). The pathway analysis module combines results from powerful pathway enrichment analysis with pathway, topology analysis to help researchers identify the most relevant pathways involved in the conditions being investigated. The analysis report was then presented graphically as well as in a detailed table. Potential biomarkers for GE efficacy were identified based on the metabolic pathway enrichment and statistics analysis.

Statistical Analysis

Statistical analysis of the integral optical density among the groups was evaluated by using the one-way analysis of variance (ANOVA), performed using SPSS 23.0 (IBM-SPSS Inc., Chicago, USA). The results were presented as mean ± standard error of mean (SEM). Orthogonal polynomial contrasts were used to test for linear and quadratic effects of GE by comparing with the NC group. Significant differences and extremely significant differences were evaluated by Tukey multiple comparisons test at p < 0.05 and p < 0.01, respectively.

As seen in Figure 1 and Table 1 , the color signals and the integral optical density of occludin and claudin-1 in the S100 and S200 groups were significantly higher than in the NC group ( p < 0.05), and NHE3 in the GE groups (GEs) was significantly higher than that of the NC group ( p < 0.05). Supplementation of GE in the diet linearly and quadratic increased the integral optical density of claudin-1 ( p < 0.01), occludin ( p < 0.01), and NHE3 ( p < 0.01) compared with the NC group.


Figure 1 . The representative figure of jejunal mucosal claudin-1 (A) , occludin (B) , zonula occludens 1 (ZO-1) (C) , and Na + /H + exchanger 3 (NHE3) (D) protein expression in different groups (immunohistochemical staining, ×200). The staining was visualized using DAB (brown), and slides were counterstained with hematoxylin ( n = 5). BC, blank control group, piglets were fed diet without supplements and ETEC challenge; NC, negative control group, piglets were fed diet without supplements and challenged by ETEC; S50, piglets were fed diet supplemented with 50 mg kg −1 of GE and challenged by ETEC; S100, piglets were fed diet supplemented with 100 mg kg −1 of GE and challenged by ETEC; S200, piglets were fed diet supplemented with 200 mg kg −1 of GE and challenged by ETEC.


Table 1 . Effect of guava extract (GE) on the integral optical density of claudin-1, occludin, zonula occludens 1 (ZO-1), and Na + /H + exchanger 3 (NHE3) in jejunal mucosa of piglets.

Analysis of Fecal Metabolomics

Using the optimal UHPLC-QTOF-MS condition described above, the representative total ion chromatograms (TICs) for fecal samples are presented in Supplementary Figure 1A . The score plots of PCA overlapped partly both in the direction of PC1 and PC2 based on the fecal samples from the NC vs. BC group ( Figure 2A1 ), S50 vs. NC group ( Figure 2B1 ), S100 vs. NC group ( Figure 2C1 ), and S200 vs. NC group ( Figure 2D1 ). Supervised PLS-DA analysis suggested that there were significant differences between two groups, indicating the distinct metabolic profiling of the NC vs. BC group ( Figure 2A2 ), S50 vs. NC group ( Figure 2B2 ), S100 vs. NC group ( Figure 2C2 ), and S200 vs. NC group ( Figure 2D2 ).


Figure 2 . Score plots of principal component analysis (PCA) models in fecal metabolomics. (A1,B1,C1,D1) represent the score plots of the PCA models (NC vs. BC group, S50 vs. NC group, S100 vs. NC group, and S200 vs. NC group, respectively), and (A2,B2,C2,D2) represent the score plots of the PLS-DA models (NC vs. BC group, S50 vs. NC group, S100 vs. NC group, and S200 vs. NC group, respectively).

Metabolic profiling in the feces was significantly changed based on the results of the NC vs. BC group, S50 vs. NC group, S100 vs. NC group, and S200 vs. NC group, respectively ( Supplementary Table 2 ). L -pipecolic acid is a unique differential metabolite between the NC group and BC group. Different metabolic pathways were enriched from the groups of S50 vs. NC, S100 vs. NC, and S200 vs. NC, respectively ( Supplementary Figure 2 ). The details of the top 4 ranked metabolic pathways and relevant differential metabolites between the BC, NC, and GEs groups are presented in Table 2 . It shows that the S50 group significantly boosted the production of 3-methoxytyramine ( p < 0.05) and decreased the production of epinephrine, normetanephrine, N -acetylserotonin, melatonin, and caffeine ( p < 0.05) compared with the NC group. The S100 group significantly upregulated the levels of biliverdin and L -pipecolic acid ( p < 0.05), and downregulated the levels of 5-aminolevulinic acid and phosphorylcholine ( p < 0.05) compared with the NC group. Moreover, the S200 group significantly downregulated the levels of uridine 5′-monophosphate (UMP), deoxycytidine monophosphate (dCMP), deoxyguanosine, and L -phenylalanine ( p < 0.05) compared with the NC group.


Table 2 . Top 4 ranked metabolic pathways and relevant differential metabolites in the feces of piglets.

Analysis of Serum Metabolomics

The UHPLC-QTOF-MS system can picture metabolic profiling of five groups with TIC ( Supplementary Figure 1B ). To investigate the global metabolic rewiring in the serum among NC, BC, S50, S100, and S200 groups, all observations were integrated and analyzed using PCA ( Figure 3 ). The score plots of PCA overlapped partly both in the direction of PC1 and PC2 based on the serum samples from the NC vs. BC group ( Figure 3A1 ) and the S50 vs. NC group ( Figure 3B1 ). The score plots of PCA from the S100 vs. NC group ( Figure 3C1 ) were separated from each other. To further explore the differences between the two groups, supervised PLS-DA was applied for chemometrics analysis ( Figure 3 ). The score plots of PLS-DA showed that the NC vs. BC group ( Figure 3A2 ), S50 vs. NC group ( Figure 3B2 ), S100 vs. NC group ( Figure 3C2 ), and S200 vs. NC group ( Figure 3D2 ) could be clearly separated, which reflected the remarkably distinct metabolic status of the serum samples among the BC, NC, S50, S100, and S200 groups. Many metabolites in the serum were significantly altered based on the results of the NC vs. BC group, S50 vs. NC group, S100 vs. NC group, and S200 vs. NC group, respectively ( Supplementary Table 3 ). Finally, different metabolic pathways were identified and further enriched as referred to as the KEGG pathway ( Supplementary Figure 3 ).


Figure 3 . Score plots of PCA models in serum metabolomics. (A1,B1,C1,D1) represent the score plots of the PCA models (NC vs. BC group, S50 vs. NC group, S100 vs. NC group, and S200 vs. NC group, respectively), and (A2,B2,C2,D2) represent the score plots of the PLS-DA models (NC vs. BC group, S50 vs. NC group, S100 vs. NC group, and S200 vs. NC group, respectively).

As shown in Table 3 , the top 4 ranked metabolic pathways between the BC, NC, and GE groups displayed characteristic differences in the serum of piglets, respectively. It is interesting that nicotinamide-adenine dinucleotide phosphate (NADP), as a node molecule, was upregulated in the NC group in comparison with the BC group and downregulated in the GE groups in comparison with the NC group ( Figure 4A ). Notably, tetrahydrofolic acid (THF) is a key node molecule affected by GE, and GE supplementation significantly upregulated the level of THF ( p < 0.05) compared with the NC group ( Figure 4B ). Additionally, considering dosage influence on the GE groups, the important top 4 ranked metabolic pathways and relevant metabolites affected also yielded dissimilar results. Especially, it suggested that GE in the S100 group significantly increased the synthesis of adenosine triphosphate (ATP), L -glutamic acid ( L -glu), and L -glutamine (Gln) ( p < 0.05) compared with the NC group. On the other hand, the NC group significantly reduced the production of thiamine pyrophosphate (TPP) ( p < 0.05) compared with the BC group. However, GE in the S200 group significantly increased the production of TPP and L -aspartic acid ( p < 0.05) compared with the NC group.


Table 3 . Top 4 ranked metabolic pathways and relevant differential metabolites in the serum of piglets.


Figure 4 . Histogram analysis of key node molecules detected in the serum of piglets: (A) nicotinamide-adenine dinucleotide phosphate (NADP); (B) tetrahydrofolic acid (THF). * p < 0.05; ** p < 0.01.

Intestinal Mucosal Barrier

In our previous study, the diarrhea incidences of piglets in the BC, NC, S50, S100, and S200 were 1.79, 21.43, 14.29, 8.93, and 7.14%, respectively. It suggested that dietary addition of GE could reduce diarrhea incidence significantly in weaned piglets challenged by ETEC ( 17 ). In general, diarrhea disease caused by ETEC infections is a major risk factor for impaired intestinal structure and barrier function of piglets. It has been reported that claudins and occludins are considered in the tight junction protein components, which primarily regulated the permeability of uncharged and charged molecules. Furthermore, ZO-1 is the adaptor protein that modulates the actin cytoskeleton ( 24 , 25 ), and NHE3 is a primary mediator of the absorptive route for Na + entering the intestinal epithelium from the lumen ( 26 ). Thus, all of them play important roles in mediating the functional integrity of the junction in epithelial and endothelial cells of the intestines ( 24 – 26 ). Our results showed that ETEC decreased the expression of epithelial tight junctions, such as claudin-1, occludin, ZO-1, and NHE3, thereby, in turn, increasing cellular permeability and disturbed the intestinal mucosal barrier. Subsequently, luminal antigens rather than bacteria may enter the lamina propria, resulting in inflammation ( 27 ). However, weaned piglets fed a diet supplementation with GE (50–200 mg kg −1 in the diet) are characterized by increased expression of claudin-1, occludin, ZO-1, and NHE3, which are crucial for the formation of a semipermeable mucosal barrier and the recovery of the barrier function of intestinal tight junctions compared with the NC group. Furthermore, previous studies also suggested that GE was rich in phenolics ( 28 ), and phenolics have a positive effect on gut health ( 29 ). Specifically, quercetin and myricetin, known as the main phenolic constituents in GE ( 17 , 30 ), have been demonstrated to enhance intestinal barrier function ( 31 ). Consequently, it is assumed that the abundant phenolics in GE exerted anti-inflammatory ( 32 ) and anti-diarrhea activity ( 15 ), and improved the intestinal barrier function and gut mucosal integrity of piglets.

Fecal Metabolomics

In mammals, L -pipecolic acid has long been recognized as a metabolite of lysine degradation ( 33 ). In this pathway, peroxisomal sarcosine oxidase (PSO) can catalyze L -pipecolic acid and oxygen to yield ( S )-2,3,4,5-tetrahydropiperidine-2-carboxylate and hydrogen peroxide (H 2 O 2 ). As seen in Figure 5 and Table 2 , it indicated that L -pipecolic acid was significantly lower in the NC compared with the BC group and suggested that ETEC might have activated the reactions mentioned above, and led to the consumption of L -pipecolic acid and the production of H 2 O 2 . H 2 O 2 accumulation can induce disruption of the intestinal epithelial barrier function by a mechanism involving phosphatidylinositol 3-kinase and c-Src kinase ( 34 , 35 ). Here, consistent with the results of immunohistochemistry, in the present study, it suggests that H 2 O 2 -induced oxidative stress in the gut might have been considered to be one of the important pathogenic mechanisms in the NC compared with the BC group, which disrupted intestinal epithelial tight junctions and barrier functions.


Figure 5 . Top 4 ranked metabolic pathways and related differential metabolites in the feces of piglets. The details of abbreviated metabolites: AANAT, serotonin N -acetyltransferase; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ASMT, acetylserotonin O -methyltransferase; ATP, adenosine triphosphate; ALAS1, 5-aminolevulinate synthase; CDA, calcium-transporting ATPase; CHKA, choline kinase alpha; COMT, catechol O -methyltransferase; DBH, dopamine beta-hydroxylase; DPYD, dihydropyrimidine dehydrogenase [NADP(+)]; DPYS, dihydropyrimidinase; GMP, guanosine 5′-monophosphate; GMPR, guanosine 5′-monophosphate oxidoreductase 1; GMPS, GMP synthase (glutamine hydrolyzing); HMOX1, heme oxygenase; IMP, inosine-5′-monophosphate; IMPDH1, inosine-5′-monophosphate dehydrogenase 1; NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NT5C2, cytosolic purine 5′-nucleotidase; PAH, phenylalanine-4-hydroxylase; PCYT1A, choline phosphate cytidylyltransferase A; PNMT, phenylethanolamine N -methyltransferase; PNP, polyribonucleotide nucleotidyltransferase; PSO, peroxisomal sarcosine oxidase; SAH, S -adenosyl- L -homocysteine; SAM, S -adenosylmethionine; TYMP, thymidine phosphorylase; UPB1, beta-ureidopropionase; UPP2, uridine phosphorylase 2; XMP, xanthosine monophosphate; AH2 and A are two generic compounds.

In addition, catecholamines are generally associated with stress events that result in high levels of Gram-negative pathogens, such as Escherichia coli ( 36 ). The metabolic results between the S50 vs. NC group suggested that dietary addition with 50 mg kg −1 GE can decrease the production of stress hormones, such as catecholamines (epinephrine and normetanephrine), and increase the production of 3-methoxytyramine (an inactive metabolite of dopamine) through tyrosine metabolism and catecholamine biosynthesis pathways and finally inhibit the growth of Escherichia coli against oxidative stress in the gut. In addition, it has been reported that caffeine can increase intracellular calcium levels through direct effects on metabolic phosphorylase-like enzyme (PHOS) regulation and calcium mobilization from the sarcoplasmic reticulum ( 37 ). Compared with the NC group, the caffeine level in the feces from the S50 group was downregulated, which may reduce the intracellular calcium content, thereby inhibiting the pathophysiological process that leads to diarrhea ( 38 ).

Based on the fecal metabolomics data of the S100 vs. NC group, it showed that 100 mg kg −1 of GE upregulated the level of biliverdin and downregulated the level of 5-aminolevulinic acid in the gut via the porphyrin metabolism pathway. This process started as the condensation of glycine and succinyl-CoA by 5-aminolevulinate synthase (ALAS) and generated 5-aminolevulinic acid. Presumably, the resulting 5-aminolevulinic acid has two fates. On one hand, it may be finally converted into biliverdin via a series of metabolic steps, leading to the accumulation of biliverdin in the gut ( Figure 5 ). The biliverdin generated in this process can protect the intestines from oxidants and inflammation ( 39 , 40 ). On the other hand, 5-aminolevulinic acid was also potentially absorbed into the blood, resulting in the high level of 5-aminolevulinic acid in the serum via glycine and serine metabolism ( Figure 6 ). Here, 5-aminolevulinic acid reduced intracellular carbon monoxide and inhibited oxidative stress and inflammation response ( 41 ). Moreover, phosphorylcholine was downregulated in the S100 group compared with the NC group, which was associated with the phosphatidylcholine and phospholipid biosynthesis pathways. It means that most of the choline of the S100 group might not be catabolized in the gut but was absorbed into the blood, and then it fluxed into betaine metabolism and was probably utilized for betaine biosynthesis ( Figure 6 ). Notably, L -pipecolic acid in the feces of the S100 group was significantly higher than that of the NC group. Conversely, L -pipecolic acid in the feces of the NC group was significantly lower than that of the BC group. Our findings suggested that 100 mg kg −1 of GE might inhibit the activity of PSO and reverse the lower levels of L -pipecolic acid caused by ETEC, which in turn prevented the production of H 2 O 2 and decreased oxidative stress level.


Figure 6 . Top 4 ranked metabolic pathways and related differential metabolites in the serum of piglets. The details of abbreviated metabolites: ACLY, ATP-citrate synthase; ADP-Rib, adenosine diphosphate ribose; AHCY, adenosylhomocysteinase; AKR1B1, aldose reductase; ALAS1, 5-aminolevulinate synthase; ALDH3A1, aldehyde dehydrogenase dimeric NADP-preferring; AMDHD1, probable imidazolonepropionase; BHMT, betaine–homocysteine S-methyltransferase 1; βine, betaine; CS, citrate synthase; DHFR, dihydrofolate reductase; DMGDH, dimethylglycine dehydrogenase, mitochondrial; F6P, fructose 6-phosphate; Fglu, N-formyl- L -glutamic acid; FPNT, formamidopyrimidine nucleoside triphosphate; 5-FTF, N5-formyl-THF; FTCD, formimidoyltransferase-cyclodeaminase; G6P, glucosamine 6-phosphate; GA, glyceric acid; GCH1, GTP cyclohydrolase 1; GCLC, glutamate–cysteine ligase catalytic subunit; GCLM, glutamate–cysteine ligase regulatory subunit; GFPT1, glutamine-fructose-6-phosphate aminotransferase; Gln, L-glutamine; Glu-Cys, glutamylcycteine; GLYCTK, glycerate kinase; GNMT, glycine N-methyltransferase; GNPNAT1, glucosamine 6-phosphate N-acetyltransferase; GPT, glutamate pyruvate transaminase; GPX1, glutathione peroxidase 1; GSSG, oxidized glutathione; GSR, glutathione reductase; GSS, glutathione synthetase; GTP, guanosine triphosphate; HAL, histidine ammonia-lyase; HDC, histidine decarboxylase; HNMT, histamine N-methyltransferase; IDPA, 4-imidazolone-5-propionic acid; L-glu, L -glutamic acid; MAOA, amine oxidase (Flavin containing) A; MAT2A, S -adenosylmethionine synthase isoform type-2; MAT2B, methionine adenosyltransferase 2 subunit beta; MDH1, malate dehydrogenase; ME1, NADP-dependent malic enzyme; MIDAC, methylimidazoleacetic acid; MIDAD, methylimidazole acetaldehyde; MTHF, 5-methyltetrahydrofolic acid; MTR, methionine synthase; N-AG6P, N-acetyl-D-glucosamine-6-phosphate; N-AG, N-acetyl- D -glucosamine; NAGK, N-acetyl-D-glucosamine kinase; PC, pyruvate carboxylase; PDHA1, pyruvate dehydrogenase E1-alpha; PDHB, pyruvate dehydrogenase E1-beta; PEMT, phosphatidylethanolamine N-methyltransferase; PGA, 3-phosphoglyceric acid; PPE, phosphatidyl-ethanolamide; PPME, phosphatidyl-N-methylethanolamide; QDPR, dihydropteridine reductase; SARDH, sarcosine dehydrogenase; SHMT1, serine hydroxymethyltransferase cytosolic; THF, tetrahydrofolic acid; TPP, thiamine pyrophosphate; UROC1, urocanate hydratase.

Based on the S200 vs. NC group, L -phenylalanine was downregulated, which is a double-edged sword. First, L -phenylalanine is not only an essential amino acid but also a precursor of tyrosine and catecholamines (including tyramine, dopamine, epinephrine, and norepinephrine), so the lower level of L -phenylalanine might decrease oxidative stress in the intestine ( 36 ). Second, the lower level of L -phenylalanine also might lead to a decrease in gut hormone secretion, including glucose-dependent insulinotropic peptide (GIP) and cholecystokinin (CCK) ( 42 ). GIP and CCK are important hormonal regulators of the ingestion, digestion, and absorption of intestinal nutrients ( 43 , 44 ). Additionally, our results indicated that UMP and dCMP were downregulated in the S200 group compared with the NC group. Both of them were involved in the pyrimidine metabolism and lactose synthesis pathways and suggested that UMP and dCMP finally may be degraded to β-alanine through the pyrimidine metabolism pathway, and then the β-alanine synthesized probably fluxed into the alanine metabolism pathway. In this pathway, alanine and glyoxylic acid can be converted into glycine and pyruvic acid via serine-pyruvate aminotransferase. Meanwhile, D -glucose probably participated in the biosynthesis of pyruvic acid, leading to the lower level of UMP in the lactose synthesis pathway. Then the pyruvic acid generated via two pathways may be absorbed into the blood and was involved in the transfer of acetyl groups into the mitochondria pathway ( Figure 6 ). Furthermore, our data revealed that the lower level of deoxyguanosine in the feces was associated with the higher level of inosine-5′-monophosphate (IMP) in the serum in the S200 group compared with the NC group ( Supplementary Table 3 ), while the higher level of IMP, as a nucleotide, may be propitious to the growth and maturation of intestinal epithelial cells and plays an important role in intestinal immunity and health ( 45 ).

Serum Metabolomics

As seen in Figure 6 and Table 3 , based on the NC vs. BC group, ETEC challenge decreases glucosamine 6-phosphate (G6P) and oxidized glutathione (GSSG) levels and increases NADP levels in the serum by affecting the glutamate and glutathione metabolism pathways, which may result in the accumulation of H 2 O 2 in the serum. On one hand, the produced H 2 O 2 cannot be reduced to water (H 2 O), which resulted in peroxide interference and cell damage through oxidation of lipids, proteins, and nucleic acids ( 46 ). On the other hand, H 2 O 2 is not a radical but is considered a reactive oxygen species, which can induce a cascade of radical reactions and inactivate pyruvate dehydrogenase (PDH) ( 47 , 48 ), leading to accumulation of TPP in the serum and meaning that pyruvic acid cannot be synthesized into acetyl-CoA, while the latter was closely associated with fatty acid biosynthesis. In addition, the lower levels of cytidine monophosphate (CMP) and deoxyuridine triphosphate (dUTP) in the NC group compared with the BC group revealed that ETEC perturbed pyrimidine metabolism, and then, it might inhibit the process of pyrimidine-related nucleotide biosynthesis.

Interestingly, based on the S50 vs. NC group, caffeine was significantly downregulated in the feces ( Table 2 ), while it was significantly upregulated in the serum ( Supplementary Table 3 ) and suggested that most caffeine can be absorbed into the blood through the intestinal mucosa. Furthermore, the high level of caffeine in the serum could lead to an increase in lipolysis, and it is usually accompanied by the accumulation of glycerol in the serum ( 49 ). Then the high level of glycerol could raise blood osmolality, and it, in turn, probably plays a favorable role in the increase in intestinal water absorption and the decrease in sodium efflux into the intestinal lumen, and finally resulted in the attenuation of secretory diarrhea caused by ETEC ( 50 ). It is worth noting that, based on the S50 vs. NC group, indoleamine 2,3-dioxygenase 1 (IDO1) or tryptophan 2,3-dioxygenase 2 (TDO2) drives tryptophan into the kynurenine pathways that produce tryptophan catabolites, such as the high level of kynurenic acid in the serum ( Supplementary Table 3 ). In this process, it is usually accompanied by the production of folic acid and L -glu, meaning that the generated folic acid and L -glu can be synthesized to THF through the folate metabolism pathway. Meanwhile, THF also was biosynthesized in the serum via two pathways, including pterine biosynthesis and histidine metabolism.

Based on the S100 vs. NC group, the higher level of L -glu supplies the amino group for the biosynthesis of other amino acids, is a substrate for glutamine and glutathione synthesis, and is the key neurotransmitter in biological systems. It revealed that after glutamine synthetase or glutaminase liver isoform (GLS2) converts L -glu into Gln, and glutamine-fructose-6-phosphate aminotransferase (GFPT1) subsequently converts Gln and fructose 6-phosphate (F6P) into L -glu and G6P, it suggested that 100 mg kg −1 of GE reversed the ETEC-induced downregulation of G6P. The higher level of G6P in the S100 group, in turn, can be converted into N -acetyl- D -glucosamine-6-phosphate ( N -AG6P) (via glucosamine 6-phosphate N -acetyltransferase) compared with the NC group. Here, downregulation of uridine diphosphate- N -acetylglucosamine and upregulation of ATP in serum indicated that most N -AG6P generated likely can be converted into N -acetyl- D -glucosamine ( N -AG) (a polysacchatide) and ATP, via N -acetyl- D -glucosamine kinase (NAGK). The resulting N -AG has confirmed its anti-inflammatory efficacy for inflammatory bowel disease ( 51 ). It is worth mentioning that betaine, which might be synthesized from choline, can be degraded via two pathways. The first pathway involves betaine metabolism. Compared with the NC group, the higher levels of S -adenosyl- L -homocysteine (SAH), L -methionine, THF, and ATP in the S100 group indicated that THF cofactors were probably used to carry and activate one-carbon units via the folate-mediated one-carbon transfer pathway, resulting in the remethylation of homocysteine to L -methionine, and the synthesis of purine nucleotides and thymidylate ( 52 ). In the second pathway, betaine can be synthesized to dimethylglycine in the methionine cycle, then the generated dimethylglycine can be converted into sarcosine and enter glycine and serine metabolism. Here, the sarcosine is synthesized via two pathways to create 5-aminolevulinic acid and serine, respectively. Of them, the formation pathway of serine was accompanied by the production of THF, whereas the production of L -methionine, purine nucleotides, and 5-aminolevulinic acid may participate in the processes of attenuated inflammatory responses and inhibited oxidative stress ( 53 – 55 ).

Furthermore, it showed that 200 mg kg −1 of GE reversed the ETEC-induced upregulation of NADP and TPP in the serum via the transfer of acetyl groups into the mitochondria and phytanic acid peroxisomal oxidation pathways and thereby participated in the production of acetyl-CoA, and the latter was related to the synthesis of fatty acids and sterols and the metabolism of many amino acids ( 56 ). Meanwhile, similar to the S50 or S100 groups, the levels of THF and L -methionine in the S200 group were also upregulated via betaine metabolism and the pterine biosynthesis pathway compared with the NC group.

It is worth noting that GE dietary addition can upregulate the level of THF and reverse the high level of NADP induced by ETEC compared with the NC group ( Figure 4 ). It suggested that THF is probably a main antioxidative force for GE indirectly ( 57 , 58 ). Meanwhile, GE downregulating the level of NADP also means that the NADP pool is probably maintained in a highly reduced state, which boosted antioxidant ability in response to oxidative damage ( 59 ).


Our study has demonstrated that dietary supplementation with 50–200 mg kg −1 of GE exhibited a positive effect on the recovery of intestinal tight junctions and barrier function of weaned piglets challenged by ETEC. Meanwhile, serum and fecal metabolomics analysis indicated that dietary GE (50, 100, and 200 mg kg −1 ) addition could reprogram energy metabolism through similar or distinct metabolic pathways and finally enhance the antioxidant ability of weaned piglets challenged by ETEC.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.

Ethics Statement

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Chinese Academy of Tropical Agricultural Sciences (Haikou, China).

Author Contributions

DW and HZ contributed to the study design. LZ analyzed the data and wrote the manuscript. DW and GH finished the animal experiments and determination. All authors reviewed and approved the final version of the manuscript.

This work was financially supported by the Central Public-Interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630032017036) and Special Project on Quality and Safety of Agricultural Products of Ministry of Agriculture of the People's Republic of China (No. 2130109).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The authors would like to thank Dr. Yabin Tang at the Shanghai Jiao Tong University School of Medicine for his help in the data analysis of metabolomics.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2021.656179/full#supplementary-material


ALAS, 5-aminolevulinate synthase; ATP, adenosine triphosphate; ACN, acetonitrile; CCK, cholecystokinin; CMP, cytidine monophosphate; dCMP, deoxycytidine monophosphate; dUTP, deoxyuridine triphosphate; ESI, electrospray ionization; ETEC, enterotoxigenic Escherichia coli ; F6P, fructose 6-phosphate; G6P, glucosamine 6-phosphate; GE, guava leaf extract; GFPT1, glutamine-fructose-6-phosphate aminotransferase; GIP, glucose-dependent insulinotropic peptide; Gln, L -glutamine; GLS2, glutaminase liver isoform; GSSG, oxidized glutathione; H 2 O 2 , hydrogen peroxide; IDO1, indoleamine 2, 3-dioxygenase 1; IMP, inosine-5′-monophosphate; L -glu, L -glutamic acid; NADP, nicotinamide-adenine dinucleotide phosphate; N -AG6P, N -acetyl- D -glucosamine-6-phosphate; NHE3, Na + /H + exchanger 3; PBS, phosphate-buffered saline; PCA, principal components analysis; PDH, pyruvate dehydrogenase; PHOS, phosphorylase-like enzymes; PLS-DA, partial least-squares discrimination analysis; PSO, peroxisomal sarcosine oxidase; SAH, S -adenosyl- L -homocysteine; TDO2, tryptophan 2,3-dioxygenase 2; THF, tetrahydrofolic acid; TICs, total ion chromatograms; TPP, thiamine pyrophosphate; UMP, uridine 5′-monophosphate; UHPLC-QTOF-MS, ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry; ZO-1, zonula occludens protein 1.

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Keywords: weaned piglets, metabolomics, enterotoxigenic Escherichia coli , guava leaf extract, intestinal barrier function

Citation: Wang D, Zhou L, Zhou H and Hou G (2021) Effects of Guava ( Psidium guajava L.) Leaf Extract on the Metabolomics of Serum and Feces in Weaned Piglets Challenged by Escherichia coli . Front. Vet. Sci. 8:656179. doi: 10.3389/fvets.2021.656179

Received: 20 January 2021; Accepted: 09 April 2021; Published: 24 May 2021.

Reviewed by:

Copyright © 2021 Wang, Zhou, Zhou and Hou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Hanlin Zhou, zhouhanlin8@163.com

† These authors have contributed equally to this work

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Journal of Ethnopharmacology

Review psidium guajava : a review of its traditional uses, phytochemistry and pharmacology.

Psidium guajava , is an important food crop and medicinal plant in tropical and subtropical countries is widely used like food and in folk medicine around of the world. This aims a comprehensive of the chemical constituents, pharmacological, and clinical uses. Different pharmacological experiments in a number of in vitro and in vivo models have been carried out. Also have been identified the medicinally important phyto-constituents. A number of metabolites in good yield and some have been shown to possess useful biological activities belonging mainly to phenolic, flavonoid , carotenoid , terpenoid and triterpene . Extracts and metabolites of this plant, particularly those from leaves and fruits possess useful pharmacological activities . A survey of the literature shows P. guajava is mainly known for its antispasmodic and antimicrobial properties in the treatment of diarrhoea and dysentery . Has also been used extensively as a hypoglycaemic agent . Many pharmacological studies have demonstrated the ability of this plant to exhibit antioxidant, hepatoprotection , anti-allergy, antimicrobial, antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, cardioactive, anticough, antidiabetic, antiinflamatory and antinociceptive activities , supporting its traditional uses. Suggest a wide range of clinical applications for the treatment of infantile rotaviral enteritis , diarrhoea and diabetes.

Psidium guajava , which is considered a native to Mexico (Rios et al., 1977) extends throughout the South America, European, Africa and Asia. Based on archaeological evidence. It has been used widely and known in Peru since pre-Columbian times. It grows in all the tropical and subtropical areas of the world, adapts to different climatic conditions but prefers dry climates (Stone, 1970). The main traditional use known is as an anti-diarrhoeal. Other reported uses include gastroenteritis, dysentery, stomach, antibacterial colic pathogenic germs of the intestine.

Its medicinal usage has been reported in indigenous system of medicines in America more than elsewhere. Psidium guajava Linn. (family Myrtaceae), is commonly called guave, goyave or goyavier in French; guave, Guavenbaum, Guayave in German; banjiro in Japanese; goiaba, goiabeiro in Portugal; araçá-goiaba, araçá-guaçú, guaiaba in Brazil; guayaba, guayabo in Español and guava in English (Killion, 2000). Psidium guajava is a small tree which is 10   m high with thin, smooth, patchy, peeling bark. Leaves are opposite, short-petiolate, the blade oval with prominent pinnate veins, 5–15   cm long. Flowers are somewhat showy, petals whitish up to 2   cm long, stamens numerous (Stone, 1970). Fruit are fleshy yellow globose to ovoid berry about 5   cm in diameter with an edible pink mesocarp containing numerous small hard white seeds. There has been a tremendous interest in this plant as evidenced by the voluminous work. Therefore, we aimed to compile an up to date and comprehensive review of Psidium guajava that covers its traditional and folk medicine uses, phytochemistry and pharmacology.

More recent ethnopharmacological studies show that Psidium guajava is used in many parts of the world for the treatment of a number of diseases, e.g. as an anti-inflammatory, for diabetes, hypertension, caries, wounds, pain relief and reducing fever (Table 1). Some of the countries with a long history of traditional medicinal use of guava include Mexico and other Central American countries including the Caribbean, Africa and Asia. Some of these uses will be outlined here.

Medicinal plants are an important element of the indigenous medical systems in Mexico (Lara and Marquez, 1996). These resources are part of their traditional knowledge. The Popoluca Indians of Veracruz rely on medicinal plants for their health care. They appear to have developed a system whereby they select and continue to use plants that they find the most effective for health care purposes. The folk use of guava has been documented in the indigenous groups of Mexican Indians, Maya, Nahuatl, Zapotec and Popoluca. A decoction of the leaves is used to cure cough. According to communities of Nahuatl and Maya origin and Popoluca of the region of the Tuxtlas, Veracruz, they use a guava leaf decoction to treat digestive suffering associated with severe diarrhoea. This is a frequent disease in rainy weather (Heinrich et al., 1998).

P guajava (Myrtaceae) is widely used in Mexico to treat gastrointestinal and respiratory disturbances and is used as an anti-inflammatory medicine (Aguilar et al., 1994). Commonly roots, bark, leaves and immature fruits, are used in the treatment of gastroenteritis, diarrhoea and dysentery. Leaves are applied on wounds, ulcers and for rheumatic pain, while they are chewed to relieve toothache (Heinrich et al., 1998). A decoction of the new shoots is taken as a febrifuge. A combined decoction of leaves and bark is given to expel the placenta after childbirth (Martínez and Barajas, 1991). A water leaf extract is used to reduce blood glucose level in diabetics. This hot tea was very common among the local people of Veracruz (Aguilar et al., 1994).

The leaf of Psidium guajava is used traditionally in South African folk medicine to manage, control, and/or treat a plethora of human ailments, including diabetes mellitus and hypertension (Ojewole, 2005, Oh et al., 2005).

Guava has been used widely in the traditional medicine of Latin America and the Caribbean in the treatment of diarrhoea and stomach-aches due to indigestion (Mejía and Rengifo, 2000, Mitchell and Ahmad, 2006a, Mitchell and Ahmad, 2006b). Treatment usually involves a decoction of the leaves, roots, and bark of the plant. It also has been used for dysentery in Panama and as an astringent in Venezuela. A decoction of the bark and leaves is also reported to be used as a bath to treat skin ailments. In Uruguay, a decoction of the leaves is used as a vaginal and uterine wash, especially in leucorrhoea (Conway, 2002). In Costa Rica, a decoction of the flower buds is considered an effective anti-inflammatory remedy (Pardo, 1999).

In Peru, it is used for gastroenteritis, dysentery, stomach pain (by acting on the pathogenic microorganisms of the intestine), indigestion, inflammations of the mouth and throat in the form of gargles (Cabieses, 1993). In some tribes of the forest (Tipis), the tender leaves are chewed to control toothaches by their weak sedative effect. Tikuna Indians use the decocted leaves or bark of guava for diseases of the gastrointestinal tract. It is also employed by the Indians of the Amazons for dysentery, sore throats, vomiting, stomach upsets, vertigo, and to regulate menstrual periods, mouth sores, bleeding gums, or used as a douche for vaginal discharge and to tighten and tone vaginal walls after childbirth. Flowers are also mashed and applied to painful eye conditions such as sun strain, conjunctivitis or eye injuries (Smith and Nigel, 1992). Guava jelly is tonic to the heart and constipation (Conway, 2002).

In the Philippines the astringent unripe fruit, the leaves, the cortex of the bark and the roots are used for washing ulcers and wounds, as an astringent, vulnerary, and for diarrhoea. Leaves and shoots are used by West Indians in febrifuge and antispasmodic baths; the dust of the leaves is used in the treatment of rheumatism, epilepsy and cholera; and guava leaves tincture is given to children suffering from convulsions (Morton, 1987).

In Latin America, Central and West Africa, and Southeast Asia, guava is considered an astringent, drying agent and a diuretic. A decoction is also recommended as a gargle for sore throats, laryngitis and swelling of the mouth, and it is used externally for skin ulcers, vaginal irritation and discharge (Mejía and Rengifo, 2000). In Mozambique, the decoction of leaves is mixed with the leaves of Abacateira cajueiro , to alleviate the flu, cough and pressed chest. In Mozambique, Argentina, Mexico and Nicaragua, guava leaves are applied externally for inflammatory diseases (Jansen and Méndez, 1990).

The use of medicinal plants by the general Chinese population is an old and still widespread practice. Psidium guajava leaves are example of the plant commonly used as popular medicine for diarrhoea which is also used as an antiseptic (Teixeira et al., 2003).

In Brazil the fruit and leaves are considered for anorexia, cholera, diarrhoea, digestive problems, dysentery, gastric insufficiency, inflamed mucous membranes, laryngitis, mouth (swelling), skin problems, sore throat, ulcers, vaginal discharge (Holetz et al., 2002). In USA guava leaf extracts that are used in various herbal formulas for a myriad of purposes; from herbal antibiotic and diarrhoea formulas to bowel health and weight loss formulas (Smith and Nigel, 1992).

Besides the medicinal uses Psidium guayava is employed as food, in carpentry, in construction of houses and in the manufacture of toys (Table 2).

These are characterized by a low content of carbohydrates (13.2%), fats (0.53%), and proteins (0.88%) and by a high-water content (84.9%), (Medina and Pagano, 2003). Food value per 100   g is: Calories 36–50   kcal, moisture 77–86   g, crude fibre 2.8–5.5   g, ash 0.43–0.7   g, calcium 9.1–17   mg, phosphorus (Conway, 2002), 17.8–30   mg, iron 0.30–0.70   mg (Iwu, 1993), vitamin A 200–400   I.U., thiamine 0.046   mg, riboflavin 0.03–0.04   mg, niacin 0.6–1.068   mg, ascorbic acid 100   mg, vitamin B3 40   I.U. (Fujita et al., 1985,

Biological activity

Scientific investigations on the medicinal properties of guava dates back to the 1940s. A summary of the findings of these studies performed is presented below.

This toxicologic study was conducted using dry leaves of Psidium guajava L. In this plant material, acute toxicologic study by the following methods: mean lethal dose LD 50 test in Swiss mice and alternative toxicology (acute toxic classes) in Wistar rats. We also made the genotoxic of 2 extracts, one of aqueous type, and the other of henaxic type in an in vitro system of short-term somatic segregation induction assay in the Aspergillus nidulans fungus and an in vivo assay of the dry drug in

Infantile rotaviral enteritis

A pilot study was carried out at the Nanfang Hospital, First Military Medical University, and Guangzhou on Psidium guajava (PG) leaf decoction for treating infantile rotaviral enteritis. Sixty-two patients of rotaviral enteritis were randomly divided into the verum group treated with PG and the control group treated with Gegen Qinlian decoction. The time for ceasing diarrhoeal, the content of Na + in blood, the content of Na + and glucose in stool, and the rate of negative conversion of human

Psidium guajava is a well-known medicinal plant that is frequently prescribed in various indigenous systems of medicine especially those of Central America and Africa. Guava extracts, traditionally prepared (infusions, decoctions, tinctures of the barks and leaves and ripe fruit) by many widely separated cultures for eons of time for various uses (Table 1) have, as summarised in this review, been shown by the application of modern scientific methods to indeed possess multiple disease

The pharmacological studies conducted on Psidium guajava indicate the immense potential of this plant in the treatment of conditions such as diarrhoeal, gastroenteritis and rotavirus enteritis, wounds, acne, dental plaque, malaria, allergies, coughs, diabetes, cardiovascular disorder, degenerative muscular diseases, inflammatory ailments including rheumatism and menstrual pain, liver diseases, cancer, etc. Not surprisingly, guava also exhibits antioxidant and anti-inflammatory effects as

Antidiabetic effects of extracts from Psidium guajava

Chemical components of the fruits of psidium guava, phytochemistry, anti-proliferative activity of essential oil extracted from thai medicinal plants on kb and p388 cell lines, cancer letter, effects on mice locomotor activity of a narcotic-like principle from psidium guajava leaves, inhibition of gastrointestinal release of acetylcholine by quercetin as a possible mode of action of psidium guajava leaf extracts in the treatment of acute diarrhoeal disease, intestinal anti-spasmodic effect of a phytodrug of psidium guajava folia in the treatment of acute diarrheic disease, anticough and antimicrobial activities of psidium guajava linn. leaf extract, journal of ethopharmacology, kinetic analysis on the sensitivity of glucose- or glyoxal-induced ldl glycation to the inhibitory effect of psidium guajava extract in a physiomimic system, inhibitory effect of some selected nutraceutic herbs on ldl glycation induced by glucose and glyoxal, medicinal plants in mexico: healers consensus and cultural importance, social science and medicine, studies on antimutagenic effect of guava ( psidium guajava ) in salmonella typhimurium, mutatation research, in vitro anti-rotavirus activity of some medicinal plants used in brazil against diarrhea, inhibition of growth, enterotoxin production, and spore formation of clostridium perfringens perfringens by extracts of medicinal plants, journal of food protection, review of the biology of quercetin and related bioflavonoids, food and chemical toxicology, vasodilator effects of quercetin in isolated rat vascular smooth muscle, european journal of pharmacology, reversal of phospholamban-induced inhibition of cardiac sarcoplasmic reticulum ca 2+ -atpase by tannin, biochemical and biophysical research communications, antibacterial and wound healing properties of methanolic extracts of some nigerian medicinal plants, triterpenoids from the leaves of psidium guajava, sos-red fluorescent protein (rfp) bioassay system for monitoring of antigenotoxic activity in plant extracts, biosensors and bioelectronics, flora medicinal indígena de méxico, guava extract ( psidium guajava ) alters the labelling of blood constituents with technetium-99m, journal of science, dual effects of quercetin on contraction in cardiac and skeletal muscle preparations, research communications in molecular pathology and pharmacology, atlas de las plantas de la medicina tradicional mexicana. ii, isolation of antimicrobial compounds from guava ( psidium guajava l.), bioscience, biotechnology and biochemistry, two new triterpenoids from the fresh leaves of psidium guajava, planta medical, chemical constituents from the leaves of psidium guajava, natural product research, effects of two medicinal plants psidium guajava l. (myrtaceae) and diospyros mespiliformis l. (ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture, journal of zhejiang university science.

Cholinergic blocking effect of quercetin

Thai journal of pharmacology.

Determination of organic acids and sugars in guajava L. cultivars by high-performance liquid chromatography

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Plants as antidiarrhoeals in medicine and diet

Identification and validation of novel genomic SSR markers for molecular characterization of guava (Psidium guajava L.)

Guava ( Psidium guajava L., family Myrtaceae ), is an important fruit crop in the tropics and subtropics across the globe. An enormous amount of diversity has been reported in Psidium cultivars and species from several countries. In the present study, 40 novel SSR markers were discovered and developed using the genome data (GCA_002914565.1) and genetic diversity analysis among 19 guava germplasm, including 11 commercially important cultivars/varieties and 8 Psidium species. Of the 40 novel SSRs, 14 were highly polymorphic and able to discriminate all the accessions examined by us. The average heterozygosity index (Hi) was found to be highest for mPg SSR26 (0.4211). Polymorphic Information Content (PIC) ranged from 0.365 to 0.487with an average of 0.410. Marker index ranged between 0.000125 ( mPg SSR26–1) and 0.015469 ( mPg SSR35). The resolving power was found to vary between 0.211 ( mPg SSR26–1) and 2.105 ( mPg SSR37). Phylogenetic analysis generated two main clades, one consisting of the Psidium species, and the other of guava varieties and 3 Psidium species. In addition be quite efficient in terms of best average heterozygosity index, marker index, Shannon diversity index, and PIC values, these novel SSRs were also able to perform cross-species amplification in other members of Myrtaceae with a cross-species transferability rate of 35.0%. These results open up interesting prospects for their use in the marker-assisted genetic improvement of guava.

Effect and mechanism of norfloxacin removal by guava leaf extract in the ZVI/H<inf>2</inf>O<inf>2</inf> system

To overcome the bottlenecks of the conventional zero-valent iron Fenton-like (ZVI/H 2 O 2 ) process, such as low reagent utilization, low applicable pH, and iron sludge contamination, guava leaf extract (GLE) was used as a green promoter to enhance ZVI/H 2 O 2 process in this study. Compared with the ZVI/H 2 O 2 system, the removal rate and k obs of norfloxacin by the ZVI/H 2 O 2 /GLE system were increased by 33.76% and 2.19 times, respectively. The experimental investigation of the mechanism showed that the attack of reactive oxygen species was the main pathway for the removal of pollutants, and three types of reactive oxygen species ( 1 O 2 , O 2 − ,·OH) generations in the ZVI/H 2 O 2 /GLE system were effectively promoted by the introduction of GLE. The reactivity improvement was mainly due to the decrease of pH. At the same time, the chelation of iron ions by GLE promoted the Fe(III)/Fe(II) cycle on the catalyst surface was also a minor mechanism to improve the reactivity. This study provides a crucial reference for the practical application of guava leaf to promote the ZVI/H 2 O 2 process in environmental pollution control.

Investigation of the chemical composition of antibacterial Psidium guajava extract and partitions against foodborne pathogens

P. guajava was partitioned into aqueous and ethyl acetate fractions and studied for its antibacterial chemical constituents. The minimum inhibitory concentrations of the aqueous and ethyl acetate partitions against Escherichia coli , Salmonella Typhimurium, and Staphylococcus aureus were found to be 0.75, 0.75, 0.15, 0.5, 0.5, and 0.125%, respectively. Using LC-MS-based chemical fingerprinting, auto MS/MS fragmentation and bioactive molecular networking, 18 compounds of interest were detected. The top 10 bioactive compounds and eight additional non-bioactive compounds known to be found in P. guajava are highlighted. We report five compounds to be identified in P. guajava for the first time. Studies have indicated P. guajava to be a plant source of antibacterial compounds that could be useful in the food industry to prevent foodborne illnesses outbreaks, reduce food spoilage, and satisfy consumer demands for less synthetic chemical usage in the food industry.

A comparative analysis of leaf essential oil profile, in vitro biological properties and in silico studies of four Indian Guava (Psidium guajava L.) cultivars, a promising source of functional food

Metabolomics and transcriptomics analyses reveal the potential molecular mechanisms of flavonoids and carotenoids in guava pulp with different colors.

To understand the forming mechanism of flavonoids and carotenoids in guava pulp with different colors. Based on our previous wide-targeted metabolome analysis, the differences of carotenoids were analyzed by targeted metabolomics, and the molecular mechanism was understood through transcriptomics analysis. The results showed that cyndin-3-O-soproposide and its possible copigments (myricetin, myricitrin, quercetin, quercitrin, etc.) were the main factors that contributed to the color difference between the pink and red guava. The accumulation of these flavonoid components was mainly due to the expression of structural genes C4H, CHS, LDOX, and transcription factors MYB and NAC. β-cryptoxanthin myristate, β-cryptoxanthin palmitate, and rubixanthin palmitate were the main carotenoids leading to the yellow pulp of the yellow cultivar. PDS, ZEP, CHYB, and transcription factors NAC were involved in the accumulation of these carotenoid components. This study provides primary data for further utilization and color regulation of guava.

Antidiarrheal activity of the extracts of Valeriana jatamansi Jones on castor oil-induced diarrhea mouse by regulating multiple signal pathways

Valeriana jatamansi Jones, a traditional medicine, is used for various medicinal purposes worldwide. This species is popular for its gastro-protective properties and has been verified to exert antidiarrheal effects. Qiuxieling mixture, an oral liquid preparation used to treat diarrhea in children in clinical practice, was extracted from V. jatamansi Jones.

Aim of the study: Although Qiuxieling mixture has a good preventive effect on diarrhea children, the disgusting smell makes it intolerable. Therefore, we extracted odorless products from V. jatamansi Jones and Qiuxieling mixture. The present study is aimed to investigate the protective effects of two ethanolic extracts of V. jatamansi Jones and Qiuxieling mixture against castor oil-induced diarrhea and their possible mechanisms in mice.

The two extracts of V. jatamansi Jones and Qiuxieling mixture were detected by HPLC. A castor oil-induced diarrheal model was used to evaluate the antidiarrheal effects. The expression of Occludin in the small intestine was measured by IHC. Western blotting and immunofluorescence were used to detect the expression of proteins related to the oxidative stress and GSDMD-mediated pyroptosis signaling pathways. ELISA was used to detect the expression of IL-6 and IL-1β in the small intestine of mice with diarrhea.

The two extracts of V. jatamansi Jones and Qiuxieling mixture dose-dependently reduced the diarrhea index and the diarrhea rate, delayed the onset of diarrhea, and decreased the weight of the intestinal content. Meanwhile, they reversed the decreased expression of Occludin and restored the activity of Na + -K + -ATPase in the intestines of diarrheal mice. In addition, they reversed the depletion of GSH, attenuated the activation of the ERK/JNK pathway, promoted the Nrf2/SOD1 signaling pathways, and decreased the release of ROS in the intestines of diarrheal mice. Moreover, they suppressed GSDMD-mediated pyroptosis by downregulating the NLRP3/caspase-1/GSDMD signaling pathway.

The two extracts of V. jatamansi Jones and Qiuxieling mixture exerted protective effects on castor oil-induced diarrhea in mice through a variety of mechanisms, including antioxidant stress, restoration of tight junctions between intestinal mucosal cells and regulation of the GSDMD-mediated pyroptosis pathway.

Psidium guajava L. and Psidium brownianum Mart ex DC.: Chemical composition and anti – Candida effect in association with fluconazole

The therapeutic combinations have been increasingly used against fungal resistance. Natural products have been evaluated in combination with pharmaceutical drugs in the search for new components able to work together in order to neutralize the multiple resistance mechanisms found in yeasts from the genus Candida . The aqueous and hydroethanolic extracts from Psidium brownianum Mart ex DC. and Psidium guajava L. species were evaluated for their potential to change the effect of commercial pharmaceutical drugs against Candida albicans and Candida tropicalis strains. The tests were performed according to the broth microdilution method. Plate readings were carried out by spectrophotometry, and the data generated the cell viability curve and IC50 of the extracts against the yeasts. A chemical analysis of all the extracts was performed for detection and characterization of the secondary metabolites. The total phenols were quantified in gallic acid eq/g of extract (GAE/g) and the phenolic composition of the extracts was determined by HPLC. Fluconazole and all extracts presented high Minimum Inhibitories Concentrations (MICs). However, when associated with the extracts at sub-inhibitory concentrations (MIC/16), fluconazole had its effect potentiated. A synergistic effect was observed in the combination of fluconazole with Psidium brownianum extracts against all Candida strains. However, for Psidium guajava extracts the synergistic effect was produced mainly against the Candida albicans LM77 and Candida tropicalis INCQS 400042 strains. The IC 50 values of fluconazole ranged from 19.22 to 68.1 μg/mL when it was used alone, but from 2.2 to 45.4 μg/mL in the presence of the extracts. The qualitative chemical characterization demonstrated the presence of phenols, flavonoids and tannins among the secondary metabolites. The concentration of total phenols ranged from 49.25 to 80.77 GAE/g in the P. brownianum extracts and from 68.06 to 82.18 GAE/g in the P. guajava extracts. Our results indicated that both P. brownianum and P. guajava extracts are effective on potentiating the effect of fluconazole, and therefore, these plants have the potential for development of new effective drugs for treating fungal infections.

Chemical composition and antioxidant activity of seven cultivars of guava ( Psidium guajava ) fruits

Cytotoxic and antioxidant constituents from the leaves of psidium guajava.

Psidium guajava (Myrtaceae) is an evergreen shrub growing extensively throughout the tropical and subtropical areas. Four new compounds, guavinoside C, D, E and F ( 1 – 3 , 10 ) were isolated from the leaves of P. guajava , along with six known ones ( 4 – 9 ). Their structures were elucidated by spectroscopic analysis. Compounds 1 , 4 and 10 showed significant cytotoxic activities on HeLa, SGC-7901 and A549 cell lines, respectively. Compounds 1 and 4 – 10 showed antioxidant activities in DPPH, ABTS and FRAP assays, and five of them ( 1 , 4 – 6 , 10 ) exhibited stronger activities than that of vitamin C.

Chemotype diversity of Psidium guajava L.

The essential oil of Psidium guajava L. has been studied for pharmacological and industrial purposes, without considering the plant's genotype regarding the heterogeneity of its composition. The present study aimed to characterize the chemotype diversity of the essential oil extracted from the leaves of 22 genotypes of P. guajava grown in two different environments in the state of Espírito Santo, Brazil, and to identify the different chemical markers present in these plants. Essential oil from the leaves of the P. guajava genotypes was extracted by hydrodistillation, and its chemical composition was analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Thirty-three compounds were identified, comprising 87.5–99.0% of the total composition, with a prevalence of sesquiterpenes in all samples. The major compounds identified consisted of (E)-trans- Caryophyllene, α -Humulene, trans -Nerolidol, β -Bisabolene, β -Bisabolol, and Hinesol, the first of which was identified as a possible chemical marker for the species. Multivariate factor analysis of the chemical composition of P. guajava oil identified three chemotypes: Commercial - PAL, SEC, PS, PET, C7, C11, and C17MI, characterized by high levels of β -Selinene, α -Selinene, Hinesol, and 14-hydroxy- epi-(E)- caryophyllene, with β -Selinene and α -Selinene as the chemical markers; C10 and C13, exhibiting high levels of Elemol, trans- Nerolidol, trans-β- Eudesmol, and ( 2Z, 6Z ) - Farnesol, which were indicated as chemical markers, and Cortibel - C1, C2, C3, C4, C5, C6, C8, C9, C12, C14, C15, C16, C17LI, which retained high levels of α -Cedrene, cis-α- Bergamotene, α- Humulene, Humulene epoxide, epi-α- Cadinol, β -Bisabolol, and α -Bisabolol, with β -Bisabolol and α -Bisabolol as the chemical markers. The use of guava genotypes with different chemotypes, that are agronomically favorable to fruit production and essential oil exploitation adds value to the crop and renders it more sustainable. Given guava crops produce large amounts of leaf biomass, resulting from successive prunings, the extraction of their essential oil, which retains commercially valuable compounds, can be feasible.

Comparative study of the volatile oil content and antimicrobial activity of Psidium guajava L. and Psidium cattleianum Sabine leaves

The chemical composition of the hydrodistilled oils of the leaves of Psidium guajava L. (guava leaf) and Psidium cattleianum Sabine (strawberry guava) was determined by GC/MS analysis to identify their chemotypes. Moreover, in vitro antimicrobial activity of these volatile oils against selected bacteria, yeast, and mycelia fungi was studied. The yield of the volatile oil hydrodistilled from the leaves of P. guajava L. and P. cattleianum Sabine was 1.6 and 2.69   g/kg on fresh weight basis, respectively. Limonene was the major identified hydrocarbon in P. guava leaves’ oil (54.70%), whereas, 1, 8-cineole was the major identified oxygenated monoterpenoid (32.14%) in common guava leaves. The foliar oil of P. cattleianum was predominated by the sesquiterpene hydrocarbon; β- caryophyllene representing 28.83% of the total oil make-up. The antibacterial activity of guava leaf oil was more pronounced against Bacillus subtilis , Staphylococcus aureus , Streptococcus faecalis , Escherichia coli , and Pseudomonas aeruginosa than that of strawberry guava leaves, while P. cattleianum showed a higher activity against ess. The MIC of the volatile oil of the leaves of P. guajava against S. aureus was 6.75   μg/ml, while that of P. cattleianum exhibited MIC value of 13.01   μg/ml against Neisseria gonorrhoeae . Results demonstrated that the volatile oil of both Psidium species showed different chemotypes. Moreover, the volatile oils of guava and strawberry guava leaves might be good candidates as antimicrobial agents.

The Potency of Guava Psidium Guajava (L.) Leaves as a Functional Immunostimulatory Ingredient

The potential of natural substances to improve the immune system has long been the subject of investigation. The purpose of this research was to study Guava ( Psidium guajava L . ) leaf extract as a functional ingredient for immunostimulant. The study used water and ethanol as solvents to obtain optimum active compounds of the extracts. The result showed that the higher the content of phenol total was found in the extract, the higher the stimulation index value was obtained for both solvents. However, the stimulation index value was not only influenced by antioxidant activity. The reason was that the type of active compound in Guava leaf extract responsible for immunostimulatory activity was probably not only polyphenolic antioxidant.


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