Ethnobotanical Review of Selected Medicinal Plants in Guam for the Treatment of Urinary Tract Ailments and Their Pharmacological Properties

: The Chamorro healers of Guam have more than a thousand years of history of using herbs and medicinal plants for the treatment of common ailments. The objective of this study is to review the bioactive compounds and pharmacological properties of medicinal plants which are used for urinary tract health by local healers. Literature searches were performed using Google Scholar, ScienceDirect, PubMed, and SpringerLink, by using several keywords, including “medicinal plants in Guam”, “traditional uses”, “bioactive compounds”, “pharmacological properties”, and “urinary tract infections”. This review highlights the traditional uses, bioactive compounds, and pharmacological properties of ﬁve medicinal plant species, namely Euphorbia hirta , Phyllanthus amarus , Premna serratifolia , Psidium guajava , and Urena lobata . Phenolics, alkaloids, terpenoids, essential oils, and polypeptides are the leading secondary metabolites reported in different plant extracts, which have been found to have signiﬁcant antimicrobial, antioxidant, anti-inﬂammatory, antidiabetic, and anticancer properties. The therapeutic claims made about medicinal plants in Guam are well supported by the literature, having similar applications and pharmacological properties in other regions of the world. These medicinal plants have a lot of unexplored potential that might be utilized to develop more potent drugs for the treatment of infectious diseases, as well as food and herbal supplements.


Introduction
Guam is the largest western Pacific tropical island of the United States and lies at 130 north latitude.The island is about 30 miles long and 4 to 12 miles wide, with a total area of 212 square miles, and is home to some 200 herbs and medicinal plants.Despite cultural adaptations associated with Spanish, American, and Asian culture, indigenous practices of healing and curing with medicinal plants by the Chamorro people of Guam have still survived in a modified form.Guam has a diverse range of plant groups, including limestone forests, wetland ecosystems, savannas, strands, and ravine forests.Many of these plant species have been used for centuries as herbal medicines and as preventative dietary supplements.These plants could be a potential source of new drugs to treat modern-day diseases, following extensive research involving the proper identification, phytochemical extraction, separation, and characterization of bioactive compounds.Medicinal plants have proven their therapeutic value for millennia and still hold out as the best options for finding active compounds.Over the past 50 years, the isolation and derivatization of numerous significant drugs from plants have revolutionized modern medicinal practice [1].
Currently, traditional healers of Guam treat minor ailments with massage, herbal medicines, and medicinal teas, as well as palai medicine that is applied to the skin [2].Traditional medicine uses a variety of plant parts, such as the leaves, stems, and roots of a single plant or a combination of several plant species.One of the most typical illnesses that indigenous healers address is urinary tract infections (UTIs).Several plant species have been identified by local healers for the preparation of herbal medicine to treat urinary tract problems [2].
Medicinal plants contain a wide variety of secondary metabolites, such as alkaloids, phenolic compounds, terpenoids, and sulfur-containing compounds, which have been found to have different pharmacological activities.Historically, bioactive compounds of plants have played a key role in drug discovery, especially for cancer and infectious diseases [3].COVID-19 had a positive impact on herbal medicine studies.Recently, there has been an increase in the search for plant-based drugs and nutritional supplements.The number of publications on bioactive natural product studies has increased more than 3-fold during the past ten years, according to a recent search using the keywords, "Natural Product Research" in the ScienceDirect database.Thus, this paper reviews the plants grown in Guam used in traditional remedies for urinary tract problems and their reported pharmacological properties.The selected medicinal plants are Euphorbia hirta, Phyllanthus amarus, Premna serratifolia, Psidium guajava, and Urena lobata (Figure 1).

Botanical Used for Urinary Tract Health in Guam
A urinary tract infection (UTI) is a bacterial infection that can affect any part of the urinary system, including the bladder, urethra, vagina, or kidneys.Common symptoms of UTIs include urinary urgency, frequency, discomfort above the pubic bone, and dysuria.UTIs are one of the most prevalent infections in women, occurring in 40% of women in the United States at some point in their lifetime [4].The urinary pathogen, Escherichia coli, causes the vast majority (75-90% of isolates) of UTIs, followed by Klebsiella pneumoniae, whereas other bacteria including Staphylococcus saprophyticus, Enterococcus faecalis, Proteus mirabilis, and Pseudomonas aeruginosa are less important [4,5].Although antibiotic medication is a successful method of treating UTIs, its side effects, particularly the emergence of resistant pathogenic strains, have increased interest in herbal therapies made from plant extracts.
Workman et al. [2] reported a list of healers in Guam who used several plant species for the preparation of herbal medicine to treat and prevent UTIs: (1) Mrs. De Jesus is one of the healers who prepared the Psidium guajava leaf infusion for a drink used to treat bladder infections.(2) Mr. Quinata and Mrs. Aguon both used the same recipe for a douche preparation which includes the leaves of E. hirta, P. guajava, and P. amarus.(3) To make vaginal douche for cleaning or after intercourse, Mr. Tan Red used the leaves of P. guajava, Typhonium cuspidatum, and P. obtusifolia.(4) To treat a vaginal infection, Mr. Tun Reyes prepared a douche mixture by using Ruellia blechum, Cassia tora, coconut oil, florida water and any tea.( 5) Mrs. Mendiola boiled the leaves of P. amarus, Artemisia vulgaris, E. hirta, P. guajava with water, which were strained and cooled before being used as a vaginal douche.( 6) Tan Maria used the leaves of R. blechum, E. hirta, P. amarus, P. guajava for douche preparation.She provided her douche to mothers after giving birth.(7) Mrs. Santos prepared douche from E. hirta, P. amarus, R. blechum, P. guajava, A. vulgaris.(8) For kidney problems, Mrs. Tan Ana prescribed drinks prepared with dried Zea mays, the leaves of Lagerstroemia speciosa, the root of Ficus prolixa, leaves of Persea americana, Hibiscus tiliaceus and Aloe vera, and coconut oil mashed, boiled, strained, and sipped like tea.
Manglona [6], another local healer of Guam, recently prepared herbal medicine for cleansing the body with the leaves, stems, and roots of 11 plant species, namely Randia cochinchinensis, Cassia occidentalis, Columbrina asiatica, Deeringia amaranthoides, Erythrina variegate, Maytenus thompsonii, Melanolepis multiglandulosa, Phymatodes scolopendria, Pandanus tectorius, and Premna obtusifolia.According to her recommendation, patients who exhibit the symptoms of painful urination, blood in the urine, kidney infections, back discomfort, and inability to completely empty their bladders can consume herbal extract at a dose of two full cups each day for seven days.One cup in the morning after waking up and one cup in evening before sleep.She prepared her herbal extract by boiling the leaves, stems, and roots of plants in a cooking pot with two gallons of water for 45 min [6].This traditional practice is still utilized quite often within the local community as an alternative to modern medicine.
Many of the herbs that are used in traditional medicine around the world have been studied in vitro and in clinical trials for the treatment of UTIs.According to Tache et al. [9], Pomegranate (Punica granatum L.), black chokeberry (Aronia melanocarpa Michx.), and cornelian cherry (Cornus mas L.) have great potential to be used for prevention or in a combined antibiotic therapy to cure UTIs.Pomegranate peel extracts, with the chemical composition of phenolics (gallic acid, caffeic acid, benzoic acid, cinnamic acid) and flavonoid compounds, have shown antibacterial activity against Enterobacteriaceae [Escherichia coli, Salmonella typhimurium and Shigella dysenteriae] and Gram-positive bacterium [Staphylococcus aureus] [10].The ripe fruit, stems, and leaf extracts of Black chokeberry contain a variety of bioactive compounds such as anthocyanins, epicatechin, quercetin, kaempferol-3-O-rutinoside, and hydroxytyrosol, which have been found to strongly inhibit the bacterial growth of E. coli in vitro [9].The authors also reported that the leaf extracts of Cornelian cherry contain iridoids, ellagic acid, and ellagic-tannins, acted as bacteriostatic agents against E. coli.Das [11] listed 36 ethnomedicinal plants used for the treatment of UTIs and 80 medicinal plants with anti-uropathogenic potential.According to her study, Vaccinium macrocarpon (cranberry) is the best-studied home remedy for UTIs.Proanthocyanins found in cranberries can inhibit bacteria from attaching to urinary tract walls, hence obstructing the progression of uropathogenesis [11].

Mechanisms of Action of Bioactive Compounds
Medicinal plants synthesize secondary metabolites in response to an infection (induced defense), which act as antibiotics against pathogenic microorganisms.Plant root exudates contain bacteriostatic compounds such as coumaric acid, 3-indol propionic acid, and methyl p-hydroxybenzoate, which can make a plant resistant to pathogens.Plants produce defense aggressive oxygen compounds such as superoxide radicals (•O 2− ) and H 2 O 2 , as well as nitrogen monoxide (NO), and enzymes, such as β-glucanases, chitinases, and proteinases, which damage the cell walls of bacteria and fungi [12].These antibacterial compounds found in herbal medicines could prevent or treat infections in humans.The antibacterial activities of these bioactive compounds are primarily mediated by two basic mechanisms: chemically interfering with the synthesis or function of essential bacterial components and/or evading the known antibacterial resistance mechanisms through (I) bacterial protein biosynthesis; (II) bacterial cell-wall biosynthesis; (III) bacterial cell membrane destruction; (IV) bacterial DNA replication and repair; and (V) inhibition of the metabolic pathway [13].The leaves of Arctostaphylos uva-ursi (bearberry) contain glycoside arbutoside; after consumption, it is first hydrolyzed in the gut to aglycone hydroquinone then carried to the kidneys and excreted in the urine.In the alkaline condition of urine, hydroquinone will be released to act as a direct antimicrobial agent [14].The leaf and berries of Juniperus communis (juniper) contain terpenoids which have antibacterial and diuretic activities [14].Proanthocyanidins, which are present in Vaccinium macrocarpon (cranberry) juice, are effective against E. coli, one of the main causes of bacteria-mediated UTIs, by preventing the bacteria from adhering to the bladder walls.As a result, the bacteria are more likely to be washed away during urination [15].Cinnamomum verum contains the essential oil, trans-cinnamaldehyde, and can prevent the growth of biofilm E. coli on urinary catheters by inhibiting the expression of key virulence genes in the bacterium [16].Isothiocyanates are an active constituent found in Armoracia rusticana (horseradish) that can block the uropathogenic E. coli from penetrating human cells [17].

Bioactive Compounds and Pharmacological Properties of Selected Medicinal Plants
Table 1 lists selected medicinal plants in Guam for urinary tract ailments and their major bioactive compounds and pharmacological properties.

Euphorbia hirta
Euphorbia hirta-commonly known as asthma-plant, barok, or pill-bearing spurge-belongs to the Euphorbiaceae family.It is one of the common annual herbaceous plants in Guam and considered as a weed that is native to tropical and subtropical regions, including Asia, Africa, and the Americas.Traditional healers of Guam have used the leaf extracts of E. hirta for an astringent douche mixture and the treatment of headaches for long a time [2].Most of its traditional uses involve taking the extract of E. hirta orally as an aqueous decoction.E. hirta has been used since ancient times as a popular medicinal herb in several parts of the world to treat urinary tract infections; to relieve gastrointestinal disorders such as diarrhea and dysentery; and to treat respiratory conditions such as asthma, coughs, bronchitis, and pneumonia.
E. hirta contains various secondary metabolites which contribute to its potential therapeutic properties.Ethanol extracts of E. hirta leaves and roots contain a diverse range of bioactive compounds, including alkaloids, glycosides, flavonoids, tannins, and phenolics [23].Aqueous, ethanol, and methanol extracts of E. hirta leaves contain tannins, saponins, steroids, terpenoids, flavonoids, triterpenoids, polyphenol, glycoside, anthocyanins, and coumarins [18].Many research investigations have thoroughly documented the biological properties of E. hirta extracts, which include antibacterial, antimalarial, antiinflammatory, antidiarrheal, and antioxidant activities (Table 1).Subramani et al. [19] reported antibacterial and antifungal activity against common oral pathogens such as S. mutans (ZOI = 11 ± 0.54 mm), Lactobacillus acidophilus (ZOI = 10 ± 0.04 mm), E. coli (ZOI = 13 ± 0.05 mm), and C. albicans (ZOI = 14 ± 0.59 mm) at a concentration of 5 mg/mL.Ethanol leaf extract exhibited significant anti-inflammatory activity compared to roots by inhibiting albumin denaturation, proteinase, and lipoxygenase activity, with 87.51% and 51.2%, followed by 97.30% and 54.21%, followed by 94.43% and 48.21%, respectively, at a concentration of 100 lg/mL [23].Sulaiman et al. [24] studied major groups of secondary metabolites of E. hirta such as phenolics, flavonoids, and sterols, which showed significant dose-dependent anticancer activity against Ehrlich-ascites-carcinoma-induced periotoneal ascites in mice.The ethanol extract of leaves exhibited higher ABTS (1338.3 ± 85.3 and 802.3 ± 91.0 mmol ascorbic acid equivalent/gram of dry extract, respectively) and superoxide anion (2014.6 ± 78.6 and 1528.0 ± 111.7 mmol ascorbic acid equivalent/gram of dry extract, respectively) scavenging abilities than the water extract [25].They also showed a remarkable antifungal effect against Fusarium oxysporum f. sp.vasinfectum, Alternaria solani, and Rhizoctonia solani.Different E. hirta Linn.extracts, including methanol, petroleum ether, chloroform, ethyl acetate, and butanol, were tested for their anticancer, antioxidant, and antibacterial activity by Tran et al. [20].Ethyl acetate extracts showed the strongest antioxidant activity compared to other extracts with DPPH radical scavenging assay and lipid peroxidation inhibition assay (IC50 = 10.33 ± 0.01 µg/mL; IC50 = 1.48 ± 0.12 µg/mL, respectively).The extracts (100 µg/mL) also showed significant inhibitory activity against the growth of lung cancer cells NCI-H460 and liver cancer cells Hep G2.The authors also proved the strongest antibacterial activity of ethyl acetate extracts, compared to other extracts, against B. subtilis, E. coli, S. aureus, P. aeruginosa, S. preumoniae, S. typhi, Vibrio cholearea, Shigella flexneri [20].In another study, Tran et al. [21] reported the antidiabetic activity of spray-dried bioactive compounds from E. hirta extracts.The spray powder was inhibited at 51.19% α-amylase at 10 mg/mL and reduced by 51% in fasting blood glucose (FBG) after 4 h of treatment.Additionally, the administration of spray powder for 15 days significantly decreased the fasting blood glucose level in streptozotocin-diabetic mice by 23.32%-compared to distilled water and the typical antidiabetic drug, acarbose, which both decreased the level by 30.87% and 16.89%, respectively [21].The antimicrobial properties of E. hyssopifolia and E. hirta, against pathogens complicit in wounds, typhoid and UTIs, were studied by Alisi and Abanobi [22].They reported the IC50 for E. hirta against S. typhi and E. coli was 99.67 µg/mL, and 165.90 µg/mL with no significant inhibition against S. aureus, implicated in typhoid fever and urinary tract infections.

Premna obtusifolia/serratifolia
Premna serratifolia is an evergreen shrub or small tree in the Lamiaceae family that can grow up to 7 m tall.It is one of the 200 species native to tropical and subtropical regions, including the Marianas.Leaf extracts of this plant have been traditionally used for the treatment of coughs, asthma, colds and flu, ear infections, hemorrhaging, and all types of illness [2], as well as urinary tract ailments [6].Khairunnisa et al. [31] listed several bioactive compounds of P. serratifolia extracts, including flavonoids, saponins, tannins, and triterpenoids/steroids, which exhibit both in vitro and in vivo biological activities, such as antioxidant, antiarthritic, anticholesterol, anti-inflammatory, antimicrobial, antihelmintic, antidiabetic, and anticancer/cytotoxic activity.The antibacterial activity of P. serratifolia essential oil against S. aureus and S. typhi showed weak inhibition wherein the diameter of the clear zone was 4.6 mm for S. aureus and 4.7 mm for S. typhi, at a concentration of 100% [32].Major bioactive compounds such as phytol (27.25%), α-humulene (14.21%), spathulenol (12.12%), 1-octen-3-ol (8.21%), eugenol (6.69%), and phenyl ethyl alcohol (5.81%) were reported by Rahman et al. [49] from Premna integrifolia Linn extracts, which displayed great potential of antibacterial activity against Sarcina lutea, B. subtilis, E. coli, Pseudomonas sp., K. pneumoniae, and Xanthomonas campestries with their respective zones of inhibition of 12.0 ± 1.2 to 22.1 ± 1.2 mm and MIC values of 62.5-250 µg mL −1 .The cytotoxic potential of the aquas root extract of P. serratifolia was analyzed via MTT assay, and was found to have an IC50 value of 1000 µg/mL after 48 h of incubation [33].The greatest antimicrobial activity was exhibited by an n-hexane fraction (MIC 10 µg/mL) of P. resinosa extract against S. aureus, E. faecalis, and Shigella flexneri and cytotoxic activity against Daoy, HepG2, and SK-MEL28 cell lines with IC50 values of 9.0, 8.5, and 13.2, respectively [50].The authors identified seven compounds for the first time, namely, quercetin, 3-methoxy quercetin, kaempferol, 3-methoxy kaempferol, myricetin 3,7,30-trimethyl ether, lupeol, and stigmasterol, which might be responsible for the anticancer, antimicrobial, and antioxidant activities of P. resinosa.Specific studies of P. serratifolia extract against uropapthogens were not found.

Extraction of Bioactive Compounds from Selected Medicinal Plants
Extraction is the essential first step in the analysis of natural products that leads to further separation, identification, and characterization.Extraction methods are classified into two main categories: conventional and non-traditional.Conventional extraction methods include maceration, percolation, decoction, reflux extraction, soxhlet extraction, and hydro/Steam distillation.Despite their widespread use, conventional procedures have significant drawbacks such as long extraction procedures, low extraction yield, reduced selectivity, degradation of heat-labile compounds, and the use of hazardous toxic organic solvents.To address the shortcomings of conventional methods, several non-conventional methods have been introduced, including supercritical fluid extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, pulsed electric field extraction, and pressurized liquid extraction [56].Different extraction methods and solvents used for extracting bioactive compounds from medicinal plants are given in Table 2.In vitro antibacterial activity for urinary tract infections [52] Leaves Maceration: ethanol, ethyl acetate, and water Antihyperglycemic and liver-protective effects [35] Urena lobata Leaves Soxhlet: methanol and distilled water Antibacterial activity against uropathogens [55] Leaves Maceration: acetone In vitro antibacterial activity [44] Maceration is the most common method used for the extraction of bioactive compounds from medicinal plants (Table 2).Fresh aerial parts of E. hirta were dried at 30 • C, then reduced to a coarse powder in a mill and extracted with ethanol via shaking for 48 h [22].A sample of the air-dried, pulverized leaves of P. amarus was cold extracted in distilled n-hexane, ethyl acetate, and methanol separately for 3 days, each by agitating and decanting for three successive extractions.Then, the extracts were filtered using Whatman filter paper, and the filtrate was evaporated using a rotatory evaporator [28].Singh et al. [33] used fresh roots for their study.After chopping the fresh root into small pieces, heating at 60 • C in 100 mL deionized water for 20 min, and after cooling, it was filtered through Whatman No.1 filter paper [33].Soxhlet extraction with methanol and distilled water as solvent was used to extract bioactive compounds from the leaves of U. lobata for a study of its antibacterial activity against uropathogens [55].

Conclusions
This review summarizes the reported bioactive compounds and pharmacological properties of seven plant species used traditionally in Guam to treat ailments of the urinary tract.Very limited research on screening phytochemicals and pharmacological activity has been reported for P. serratifolia.Other species such as E. hirta, P. amarus, U. lobata, and P. guajava have been found to be effective against E. coli strains, which cause the majority of UTIs.The antibacterial properties of plant extracts, which include tannins, saponins, steroids, terpenoids, flavonoids, triterpenoids, polyphenols, glycosides, anthocyanins, and coumarins, are thought to be responsible for the traditional treatment of UTIs.However, none of these Guam-grown plant species have been investigated for their phytochemical components and pharmacological properties yet.To identify all potential bioactive compounds responsible for pharmaceutical activity and their mechanisms, it is necessary to screen medicinal plants for therapeutic potential.Additional molecular-level research should also be performed.The isolation and characterization of bioactive compounds, the evaluation of their clinical safety, and the validation of their traditional uses may offer new opportunities in scientific research for the discovery of new drugs and the development of herbal supplements.

Figure 1 .
Figure 1.(a-e): Photograph of selected medicinal plants in Guam for the treatment of urinary tract ailments.Figure 1. (a-e): Photograph of selected medicinal plants in Guam for the treatment of urinary tract ailments.

Table 1 .
Literature-based proof of phytochemicals and pharmacological activities of selected herbs and medicinal plants in Guam.

Table 2 .
Methods and solvents used for extracting bioactive compounds from medicinal plants.