A Review of the Phytochemistry and Pharmacology of Phyllanthus urinaria L.

The genus Phyllanthus (L.) is one of the most important groups of plants belonging to the Phyllantaceae family. Phyllanthus urinaria (L.) is an annual perennial herbal species found in tropical Asia, America, China, and the Indian Ocean islands. P. urinaria is used in folk medicine as a cure to treat jaundice, diabetes, malaria, and liver diseases. This review provides traditional knowledge, phytochemistry, and biological activities of P. urinaria. The literature reviewed for this article was obtained from the Web of Science, SciFinder, PubMed, ScienceDirect, and Google Scholar journal papers published prior to December 2017. Phytochemical investigations reveal that the plant is a rich source of lignans, tannins, flavonoids, phenolics, terpenoids, and other secondary metabolites. Pharmacological activities include anticancer, hepatoprotective, antidiabetic, antimicrobial, and cardioprotective effects. Thus, this present review summarizes the phytochemical constituents and their biological activities including biological studies on various crude extracts and fractions both in vitro and in vivo, and on clinical trial information about P. urinaria. This review compiles 93 naturally occurring compounds from P. urinaria along with their structures and pharmacological activities. The review is expected to stimulate further research on P. urinaria, and its pharmacological potential to yield novel therapeutic agents.


INTRODUCTION
Traditional or indigenous medicine denotes medical practices developed by local ethnic people using natural herbs. Different world locations have their own history of traditional medicine. For example, Ayurveda medicine originated from Southeast Asia, Unani medicine originated from Arab countries in the Middle East, and acupuncture and traditional Chinese medicine (TCM) originated from China (Tao et al., 2014). Traditionally herbal medicines are used in folk medicine for the treatment of various health complications including inflammatory, cancerous, diabetic, hypertensive, and cardiovascular diseases (Tao et al., 2014). Medicinal plants are rich sources for new drug discovery as evidenced by some recent drugs that are from plant-derived compounds/derivatives (Harvey et al., 2015). For example, success using classic traditional medicine includes salicylic acid and artemisinin, possibly the most effective medicinal natural products ever found. The use of traditional medicinal data in the drug discovery process results in new therapeutics, and identifies leads that undergo clinical trials (Harvey et al., 2015). In general, it is believed that traditional medicines are safe and harmless as compared with modern drugs although this is seldom rigorously tested. Indications that the natural product extracts are effective against a particular pathological condition are based on the literature and do not imply that the effect has been proven using double blind studies with placebos (Izzo et al., 2016). The modern approach has the goal to establish evidence-based use of traditional medicines, both locally and globally.

Phyllanthus urinaria
The genus Phyllanthus (L.) belongs to a family of flowering plants Phyllanthaceae and consists of more than 1000 species widely distributed in various parts of the world (Mao et al., 2016). The species of this genus including trees, herbs and shrubs that are pharmacologically valuable as they contain various bioactive compounds (Calixto et al., 1998;Mao et al., 2016). Previous scientific data indicate that more than 500 chemical compounds (phytochemicals) have been isolated from species of the genus Phyllanthus (Mao et al., 2016). It is interesting to note that crude extracts obtained from species of Phyllanthus have inhibitory effects on the hepatitis B virus (HBV). Previous reviews broadly highlight the biological activities of Phyllanthus species, mostly from P. amurus Schum. & Thonn., P. emblica L. or P. niruri L. (Calixto et al., 1998;Mao et al., 2016;Kaur et al., 2017;Tewari et al., 2017;Yadav et al., 2017). However, there is no specific and detailed review of P. urinaria. To provide scientific proof for P. urinaria ethnopharmacological and traditional uses, recent scientific studies focus on its chemical constituents and their biological properties. Therefore, this review provides information about P. urinaria including comprehensive information about the traditional use of P. urinaria, its phytochemicals and their biological activities. It also includes biological studies both in vitro and in vivo on various extracts of P. urinaria, analysis of pure compounds and clinical trial information.

METHODOLOGY
The literature for this review was collected from various search engines and databases including Scifinder, Web of Science, PubMed, Google Scholar, and ScienceDirect. We considered the literature published prior to December 2017 on ethnopharmacological uses, pharmacology of extracts, and isolated pure compounds from different parts of P. urinaria. The search terms "Phyllanthus urinaria," or "P. urinaria extract, " or "P. urinaria compound" were used with no exact time limit. Potential full-texts of eligible papers were identified. All articles with title/abstract were included and no language restrictions applied. All relevant references were checked for additional and unpublished citations.

MEDICINAL USES OF P. urinaria: TRADITIONAL KNOWLEDGE
Traditionally, the whole plant, roots, fruits, and leaves of P. urinaria is used for the treatment of various complications in different regions of the world. In particular, the Chinese and Indian traditional medicine system documents different applications of parts of this plant as remedies for various health complications. For example, in traditional Chinese medicine (TCM), decoction of the whole plant of P. urinaria (Chinese name: Yexiazhu) can clear heat-toxin and remove dampness so it is employed to treat jaundice, enteritis, diarrhea, and dropsy (Xia, 1997). The TCM prescription, named "yexiazhu capsule, " claims to cure hepatitis B (Xia, 1997). In India, P. urinaria is considered a very good diuretic, and the crushed plant is used as a fish poison (Bharali et al., 2003). In Taiwan, decoction of young shoots or roots of P. urinaria is traditionally used to treat contagious hepatitis, acute conjunctivitis, diarrhea, edema, dysentery etc. . In Thailand, P. amarus, P. virgatus G. Forst., and P. urinaria share the name "look tai bai"; all of these plants are used to treat gonorrhea, jaundice, diabetes, and liver disease (Suthienkul et al., 1993;Chudapongse et al., 2010). In Malaysia, the juice is applied to stimulate children's appetite and to wash their tongues (Jantan et al., 2014). In Papua New Guinea, an extract is used as a febrifuge. In Brunei, a leaf poultice is applied with coconut milk to treat smallpox. In Cambodia, P. urinaria is used against malaria. The pills prepared from equal amounts of P. urinaria leaves and black pepper are beneficial for malarial fever (Hout et al., 2006). In Ghana, a decoction is employed to treat dysentery and in the Solomon Islands, the leaves are used to relieve pain in the chest (Agyare et al., 2014). In Madagascar, stem or leaf infusions are used to treat bronchitis and asthma (Calixto et al., 1998). In South America, a decoction is used for the treatment of kidney stones (Hout et al., 2006). Besides conventional usage, modern day scientific investigations have now confirmed pharmacological properties of P. urinaria. These previous studies suggest that P. urinaria is an effective medicinal remedy to treat and prevent a wide range of disorders.

Anticancer Activities
Epidemiological and experimental studies suggest that medicinal herbs have great potential in the management of different types of cancers including lung, breast, colon, liver, prostate, skin, and ovarian carcinomas. In this connection, medicinal plant extracts, and their purified compounds (phytochemicals) have significant growth inhibitory potential against various types of cancer cells in vitro as well as in vivo (Harvey et al., 2015). Although P. urinaria preparations traditionally are used as an alternative medicine for various cancers, there is little scientific evidence available about the use of P. urinaria as an anticancer agent (Table 1). Reported scientific data indicate that the anticancer signaling mechanism of P. urinaria extracts is through induction of apoptosis. Table 1 summarizes the inhibitory potential of P. urinaria extracts against various types of cancer cells. An aqueous extract obtained from the whole P. urinaria plant has growth inhibitory activity in different types of cancer cells including hepatoma, leukemia, and fibrosarcoma through induction of apoptosis; normal endothelial cell lines and liver cells are not affected (Huang et al., 2003(Huang et al., , 2004a. The aqueous extract reduces proliferation of Lewis lung carcinoma cells and human myeloid leukemia cells (HL-60 cells) in a dose-and time-dependent manner, without Methanolic extract from aerial parts anti-angiogenic against rat aortic vascular growth Ng et al., 2010 Ethanolic extract from whole plant Oral administration of P. urinaria extract attenuated the acetaminophen induced hepatotoxicity, and inhibition of cytochrome P450 CYP2E1 enzyme in mice Hau et al., 2009 Ethanolic extract from whole plant Protected cardiac H9c2 cells against doxorubicin-induced by influencing the nuclear localization of glutathione-S transferase Pi without affecting enzymatic activity. Chularojmontri et al., 2009 Chloroform and methanol extracts from whole plant Inhibited Helicobacter pylori, and its adhesion and invasion to AGS cells Lai et al., 2008 480 mg Korean P. urinaria extract capsule Alleviated the MCD-induced nutritional steatohepatitis through reduced oxidative stress, inflammation, and lipid accumulation Shen et al., 2008 Phyllanthus urinaria extract In vivo promote the N-cadherin expression in the testis tissues disrupted by nitrogen mustard (HN2) Zhang et al., 2008 Aqueous extract from whole plant In vitro antiplasmodial activity Hout et al., 2006 Methanolic extract from whole plant In vivo hepatoprotection against CCl 4 -induced liver damage Lee et al., 2006 Ethanolic extract from aerial part Antioxidative and Cardioprotective Chularojmontri et al., 2005 (Continued) Frontiers in Pharmacology | www.frontiersin.org Acetone, ethanol and methanol extracts from whole plant Inhibited HSV-2 but not HSV-1 infection Yang et al., 2005 Aqueous extract from whole plant In vitro growth cell inhibition in hepatoma, leukemia, fibrosarcoma and HUVEC cells Huang et al., 2003Huang et al., , 2004aHuang et al., ,b, 2006 A fraction containing 60% corilagin In vivo antithrombosis due to its inhibition of platelet-neutrophil adhesion. Shen et al., 2004 Hydro-alcoholic extract from whole plant Chemopreventive property against 7,12-dimethylbenz-anthracene (DMBA)-induced skin papillomagenesis in mice. Bharali et al., 2003 Hydro-alcoholic extract of stems, leaves and roots Caused a graded relaxation in guinea-pig trachea (GPT) pre-contracted by carbachol. Paulino et al., 1996a Hydro-alcoholic extract of stems, leaves and roots Caused graded contraction in GPT modulated by the epithelium, depends on the release of a cyclo-oxygenase metabolite, and relies largely upon an extracellular Ca 2+ influx Paulino et al., 1996b Hydro-alcoholic extract of stems, leaves and roots Antinociceptive effect in mice Santos et al., 1995 Hydroalcoholic extract substance P and substance P methyl ester Caused graded contractions in the guinea-pig urinary bladder Dias et al., 1995 50% methanolic extract from whole plant Oral administration (30 mg/kg) decreased the blood glucose levels Higashino et al., 1992 affecting the normal cells (Huang et al., 2003). Growth inhibition of HL-60 cells is associated with induction of the apoptosis signaling pathway and Fas receptor/ligand expression in CD95 cells (Huang et al., 2004b). Additionally, aqueous extracts of P. urinaria affected the human umbilical cord endothelial cells (HUVEC) by reduced blood vessel density, matrix induced tube formation, and cell migration (Huang et al., 2006). The aqueous and methanolic extracts obtained from the whole plant of P. urinaria inhibits metastasis of breast carcinoma cells (MCF-7) through extracellular signal-related kinase (ERK) and hypoxia pathways (Lee et al., 2011(Lee et al., , 2016). An aqueous extract obtained from the whole P. urinaria plant cause cytotoxic effects in various types of cancer cells by induction of DNA fragmentation and cell apoptosis along with increased caspase-3 activity and reduced telomerase activity (Huang et al., 2009(Huang et al., , 2010. It is reported that both aqueous and methanolic extracts of P. urinaria whole plant inhibit proliferation, metastasis and angiogenesis in a human melanoma (MeWo) cancer cell line through MAPKs, Myc/Max, NFκB, and hypoxia pathways (Tang et al., 2010(Tang et al., , 2014. Both aqueous and methanolic extracts of P. urinaria whole plant inhibit A549 cell metastasis by suppressed invasion and migration of A549 cells through the ERK1/2 and hypoxia signaling pathways (Lee et al., 2013b). The hot water extract from whole plants of P. urinaria induces apoptosis in human osteosarcoma 143B cells through the Fas receptor/ligand expression pathway (Wu et al., 2012). The same extract inhibits invasion and migration of another osteosarcoma cell line, Saos-2 cells through the ERK and Akt signaling pathways (Lu et al., 2013). Methanol extracts of P. urinaria aerial parts has anti-angiogenic properties against rat aortic vascular growth (Ng et al., 2010). The matrix metalloproteinases (MMPs) promote the prevention of metastasis of cancer cells. P. urinaria extracts inhibit the invasion and migration of highly metastatic A549 and Lewis lung carcinoma (LLC) cells through decreased expression of matrix MMP-2 and MMP-9, as well as transcription of MMP-2 mRNA, suggesting suppression of the function of MMPs by extracts [ethanol/water (1:1)] obtained from P. urinaria leaves (Tseng et al., 2012). These effects may relate to the presence of several cytotoxic and anticancer compounds in P. urinaria extracts. Therefore, further studies require identification of the responsible compounds for the observed anticancer activity. The results of the above studies validate the traditional claim of the anticancer activity of P. urinaria, and thus it might serve as a potential source of potent anticancer agents. The majority of previous scientific reports focus on growth inhibitory potential of P. urinaria extracts in various cancer cell lines in vitro. An aqueous P. urinaria whole plant extract has anti-angiogenesis and reduced tumor growth in Lewis lung carcinoma in vivo (Huang et al., 2003(Huang et al., , 2006. Oral administration of an aqueous P. urinaria extract inhibits human osteosarcoma xenograft growth in mice through modulation of the mitochondrial fission/fusion machinery (Huang et al., 2014(Huang et al., , 2016. Although the cytotoxic and anticancer activities of P. urinaria extracts seem promising from the reported studies, the lack of toxicity studies with appropriate normal cells, and lack of comparison with positive control drugs further restricts the current knowledge on P. urinaria as an anticancer agent.

Hepatoprotective and Antioxidant Action of P. urinaria
Liver damage can be caused by hepatitis virus infection, poor eating habits, heavy metal intoxication, alcohol intake or obstruction of the biliary tract (Zhong et al., 2013). Recent studies indicate that naturally derived products have significant hepatoprotective properties through their antioxidant, antiinflammatory and anticancer properties (Ali et al., 2018). Chronic hepatitis B is a major problem of worldwide concern (Tang et al., 2018). The traditional use of P. urinaria as therapy for virus caused-hepatitis suggests that this plant species is an antiviral agent (Ji et al., 1993;Wang et al., 1994;Zhou et al., 1997;Peng et al., 2006;Liu et al., 2008). Previous scientific data also indicate that P. urinaria has potential for the treatment of liver diseases (Tables 1, 2). For example, the methanol, acetone and ethanol extracts of P. urinaria inhibit Herpes simplex virus (HSV)-2 infection in vitro (Yang et al., 2005). Methanolic extracts of P. urinaria whole plant inhibit CCl 4 -induced acute liver damage through modulation of serum glutamate-pyruvatetransaminase and glutathione peroxidase in vivo Lee et al., 2006). These results are supported by a recent study in vivo indicating that P. urinaria attenuates CCl 4induced hepatotoxicity by regulation of L-carnitine, taurocholic acid, and amino acid metabolisms (Guo et al., 2017). Acetone extracts from whole plant of P. urinaria inhibit Hepatitis C virus infection in vitro (Chung et al., 2016). An aqueous extract from dried leaves of P. urinaria inhibits HBsAg, and HBcAg secretion and Hepatitis B virus (HBV) DNA synthesis in HBV wild type and LMV-resistant-infected HepG2 cells via the COX-2 and IL-6 signaling pathways (Jung et al., 2015). An aqueous P. urinaria extract inhibits HBV replication and expression in a HBV transient transfection model in vitro (Wu et al., 2015). Sharma et al. (2011) reported that a methanolic extract of P. urinaria whole plant protects the Hep G2 cell line against tert-butyl hydroxide (t-BH)-induced cytotoxicity. An ethanolic extract of P. urinaria whole plant attenuates the acetaminophen-induced hepatotoxicity and inhibition of the cytochrome P450 CYP2E1 enzyme in mice (Hau et al., 2009). A P. urinaria extract (480 mg capsule), contains corilagin, flavonoids and polysaccharides; it attenuates steatohepatitis in cultured hepatocytes in vitro and in methionine-and-cholinedeficient diet-fed mice in vivo (Shen et al., 2008). P. urinaria has anti-steatohepatitis effects through its anti-inflammatory activity (reduced TNF-α and IL-6 production through JNK and NF-κB pathways), induction of fatty acid oxidation (upregulation of CYP4a10 and suppression of C/EBPβ), and antioxidant properties (reduced CYP2e1 expression) (Shen et al., 2008). Xu et al. (2007) indicate that a 60% aqueous acetone extract from the whole P. urinaria plant has antioxidant activity in the 1,1-diphenyl-2-picrydydrazyl (DPPH)-radical assay with an SC 50 (50%-scavenging concentrations) value of 14.3 mg/mL). The presence of flavonoids, tannins and phenolic compounds in P. urinaria suggest that they contribute the observed antioxidant activity. P. urinaria extracts have anti-nociceptive effects in mice (Santos et al., 1995(Santos et al., , 1999, liver cell protection against CCl 4injury (Zhou et al., 1997), relaxation of guinea pig trachea (Paulino et al., 1996a,b) and induction of the contractile response in urinary bladder (Dias et al., 1995). The aqueous extract from whole plant of P. urinaria inhibit HBV DNA polymerase inhibition in vitro (Chen et al., 1995). The cocktail extract from whole plant of P. urinaria help to reduce activity of dengue virus-2 (Lee et al., 2013a). The ethyl acetate and n-butanol fractions from a MeOH extract of P. urinaria exhibit antiviral activity against enterovirus 71 (EV71), coxsackie virus A16, and CA16 (Yeo et al., 2015). Aqueous extracts of P. urinaria whole plant have antiviral activity against herpes simplex virus type-1 (HSV-1) and HSV-2 with selective index (SI) value >33.6 [(SI = 50% cytotoxic concentration (CC 50 )/ half inhibitory concentration (IC 50 )]; the P. urinaria extract may act against the early infection stage and the replication stage in cells in vitro (Tan et al., 2013). The ability of P. urinaria to inhibit the replication of HBV in vivo and in vitro indicates its consideration as a potential therapeutic for HBV infection.

Anti-Diabetic Effects of P. urinaria
The chronic metabolic disorder, diabetes mellitus is caused by deficiency of insulin secretion and/or decreased response of organs to insulin (Owens et al., 2017). The insulin resistance in type-2 diabetes is normally followed by β-cell dysfunction that causes hyperglycemia (Owens et al., 2017). Commercial drugs are expensive and usually have undesired side effects and toxicity (Owens et al., 2017). Therefore, there is a need to develop an alternative treatment for diabetes. Recent studies focus on the antidiabetic potential of natural products including antihypoglycemic or anti-glycation properties and on α-glucosidase inhibition. The enzyme, α-glucosidase cleaves carbohydrates into glucose and elevates the blood glucose level. Therefore, αglucosidase inhibitors are considered as antidiabetic agents for type-2 diabetes (Dash et al., 2018). The use of natural products as α-glucosidase inhibitors has gained interest because they do not induce toxicity or negative symptoms for the liver, kidney, and gastrointestinal system. Ethanol and water extracts obtained from whole plant of P. urinaria inhibit α-glucosidase with IC 50 values of 39.7 ± 9.7 and 14.6 ± 4.6 µg/mL, respectively (Trinh et al., 2016). A 50% aqueous methanol-soluble extract of the leaves of P. urinaria inhibits porcine pancreatic amylase (Gunawan-Puteri et al., 2012). Oral administration of a 50% methanol extract (30 mg/kg) of P. urinaria whole plant decreases blood glucose levels (BGL) by 24%, after three h. (Higashino et al., 1992). The P. urinaria extract fractionated with n-butanol reduced the BGL by 23 and 39% at concentration of 10 and 30 mg/kg, respectively. The 30 mg/kg treatment completely abolished the enhanced BGL (Higashino et al., 1992). The findings emanating from these studies indicated the potential of P. urinaria as an antidiabetic agent (Table 1), and this might be explored in the development of new pharmaceuticals. However, the antidiabetic potential of P. urinaria needs further study including protection of pancreatic β-cells against oxidative damage and insulin secretion and postprandial blood glucose levels in models in vitro and in vivo.

Antimicrobial Activity of P. urinaria
Antimicrobial activity of P. urinaria is indicated in Table 1. It known that Helicobacter pylori is resistant to most antibiotics, but P. urinaria preparations have antimicrobial activity against this bacterium. Chloroform and methanol extracts of P. urinaria whole plant have superior anti-H. pylori activity compared with its pure compounds . The chloroform extract potently inhibits H. pylori adhesion and invasion of gastric epithelial AGS cells, whereas the methanol extract has a moderate effect. The chloroform extract attenuates H. pylori-induced NF-κB activation with subsequent release of IL-8 . The anti-plasmodial activity in vitro of aqueous, methanol, and dichloromethane extracts of P. urinaria whole plant against a chloroquine-resistant Plasmodium falciparum strain (W2) indicates that the methanolic extract of P. urinaria is as active       Hout et al., 2006). The methanolic extract of P. urinaria whole plant also has potent anti-malarial activity toward chloroquinesensitive (CQS) strains of P. falciparum with an IC 50 = 4.1 µg/mL (Haslinda et al., 2015). The mechanism behind the antimicrobial action of P. urinaria extracts is associated with the presence of metabolites including phyllanthin, phyltetralin, rutin, quercetin, trimethyl-3,4-dehydrochebulate and methyl brevifolincarboxylate ( Table 2). These compounds present in P. urinaria extracts may interact with the proteins present in the microbial cell membrane to form stable water-soluble complexes, resulting in microbial cell death.

Cardioprotective Effects of P. urinaria
In recent years, there is interest in naturally occurring cardioprotective agents that may lack side effects. Herbal products are widely used among patients with cardiovascular (CV) diseases, and patients often combine herbal products with CV medications. Extracts of P. urinaria have cardio-protective effects in vitro in streptozotocin-induced diabetic rats ( Table 1).
The ethanolic extract of P. urinaria whole plant has antioxidant and cardioprotective effects against doxorubicin toxicity in H9C2 cardiac myoblasts (Chularojmontri et al., 2009). The ethanolic extract from the aerial parts of P. urinaria increase the activity of catalase/superoxide dismutase, increase total glutathione concentration and inhibit lipid peroxidation. The extract induces apoptosis in H9c2 cells through the NF-κB and caspase-3 activation signaling pathway (Chularojmontri et al., 2005(Chularojmontri et al., , 2009). These studies indicate that crude extracts of P. urinaria have cardioprotective potential and might lead to promising agents for therapeutic development to treat cardiac complications.

Other Activities of P. urinaria Extracts
Ethanol extracts of P. urinaria whole plant stimulate antiarthritic activity in vitro (Buddhachat et al., 2017). The methanol extract obtained from whole plant of P. urinaria increases phagocytosis of human phagocytes (Yuandani et al., 2013). Extracts of P. urinaria promote N-cadherin expression in vivo in the testicular tissues disrupted by nitrogen mustard (Zhang et al., 2008). The hydro-alcoholic extract of P. urinaria whole plant prevents 7,12-dimethylbenz(a)anthracene (DMBA)induced skin papillomagenesis in vivo (Bharali et al., 2003). A fraction containing 60% corilagin obtained from whole plant of P. urinaria has antithrombosis activity through inhibition of platelet-neutrophil adhesion (Shen et al., 2004).

PHYTOCHEMICAL CONSTITUENTS OF P. urinaria
Traditionally human populations consume herbs and their extracts. Many modern medicines use standardized plant extracts as active constituents. Various phytochemical groups have been isolated and identified from P. urinaria by chromatographic techniques. These constituents include lignans, tannins, flavonoids, phenolic acids, terpenoids, and other compounds ( Table 2). To date, 93 compounds have been identified and structurally elucidated from the extracts of P. urinaria including 22 lignans, 16 tannins, 12 flavonoids, 21 phenolics, 13 terpenoids, and other secondary metabolites ( Table 2). Typical structures of isolated constituents from P. urinaria are shown in Figures 1-6. The chemical profiles of P. urinaria may vary with the geographical production region, plant organs used and extraction procedure. Lignans and tannins exhibit various activities and are considered the major biological active compounds of P. urinaria Zhong et al., 1998;Liu et al., 1999;Giridharan et al., 2002;Yang et al., 2007a,b;Fang et al., 2008;Cheng et al., 2011;Huang et al., 2011). Corilagin, geraniin, and gallic acid are the three most prevalent compounds in P. urinaria, and pharmacological researches mainly focus on phyllanthin, hypophyllanthin, corilagin, geraniin, brevifolin and its derivatives, and rutin. The list of compound names and their biological activities are presented in Table 2.

Flavonoids
Flavonoids, are a group of natural substances consisting of two aromatic rings joined by a three carbon-oxygenated heterocycle. These are the most numerous group of polyphenolic phytonutrients (plant chemicals) and are found in most fruits and vegetables. Flavonoids have various pharmacological activities including anticancer, anti-inflammatory, antioxidant, anti-diabetic, and antiviral activites through various cellsignaling pathways (Mozaffarian and Wu, 2018). Most of the flavonoids reported from P. urinaria are in the flavonol and glycoside form (Nara et al., 1977) (Table 2). From the ethanolic extract of P. urinaria, two new acetylated flavonoid glycosides 47, 48, along with the known isolates, quercetin (42) and quercetin 3-O-α-L-rhamnopyranoside (49) have been isolated (Wu et al., 2013). A new flavone sulfonic acid, urinariaflavone (44) was isolated from the methanolic extract of P. urinaria (Thanh et al., 2014). The isolated flavonoids from P. urinaria showed antioxidant, anti-inflammatory, anticancer, and anti-H. pylori etc., activities ( Table 2).

Terpenoids
Terpenoids (or isoprenoids) are compounds derived from one or more five-carbon isoprene units. These compounds represent the most diverse class of beneficial phytochemicals with anticancer, anti-cardiovascular, anti-Alzheimers, and antimalarial activities. The terpenoids such as taxol, artemisinin, and ginkgolides have therapeutic effects on a variety of diseases (Cho et al., 2017). A number of terpenoids (13 compounds) including three triterpenoids (72-74), two diterpenoids (75 and 76), two sesquiterpenes (77 and 78), two monoterpenes (79 and 80), and four sterols (81-84) have been isolated from the extracts of P. urinaria ( Table 2). Fractionation of the acetone extract from P. urinaria resulted in the isolation of a monoterpenoid lactone, loliolide (80) that has anti-HCV activity through inactivation of virus particles, revocation of HCV attachment and reduced viral fusion (Chung et al., 2016). The pentacyclic oleanane-type triterpenoid β-amyrin (72) has anti-inflammatory, anti-nociceptive, antimicrobial, and anti-apoptotic activities (Askari et al., 2018). Oleanolic acid (74) and its derivatives have therapeutic potential against various types of cancers in vitro and in vivo (Ayeleso et al., 2017).

CLINICAL TRIALS OF P. urinaria PREPARATIONS
It known that clinical trials are required for any new compound to enter into the market. Table 3 summarizes the important clinical trials of P. urinaria. In China, 140 chronic hepatitis B patients treated for two years have a recovery rate expressed as the index of HBV-DNA and HBeAg of 88.2% and 52.5%, respectively. Once the treatment is stopped, the recurrence rate is 10.4-13.4% respectively . Tong et al. (2014) reports that  compound in capsule of P. urinaria L. suppresses development of hepatocellular carcinoma (HCC) through an improved immune system, reversion of liver fibrosis, blockage of the induced hepatocarcinoma cell cycle and inhibition of angiogenesis. The HBV-DNA levels decrease ≥2 log in 22.2% (10/45) of patients in the treatment group compared with the control group at 5.0% (2/40). The number of antibodies that test positive in the treated group is lower (1.08 ± 1.01) after the treatment period of 24 months compared with the control group (2.11 ± 1.12) (Tong et al., 2014). The anti-URG11 (33/52) and anti-URG19 (31/52) in both treated and control groups are over 60% at base line. After the treatment period of two years, in the treated group the levels of anti-URG11 and anti-URG19 decreased to 48.1% (25/52) and 46.2% (24/52), whereas in the control group the

Sample
Result Reference Compound P. urinaria L (CPUL) CPUL prevented or delayed in the development of HBV-associated cirrhosis to HCC through improved immune system, revert liver fibrosis, induced hepatocarcinoma cell cycle block and inhibited angiogenesis. Tong et al., 2014 400 mg of P. urinaria tablet Phyllanthus is not superior to placebo in improving NAFLD activity score in NASH patients Wong et al., 2013 Phyllanthus Pill After treatment with P. urinaria capsule for 3 months or 2 years, the recovery rate in the index of HBV-DNA and HBeAg was 88.2% and 52.5%, respectively. Cheng et al., 2009 P. urinaria extract Received P. urinaria 1, 2 and 3g three times daily for 6 months, there was no difference in log10 [HBV DNA] reduction, HBeAg seroconversion and ALT normalization, suggested P. urinaria had no demonstrable anti-viral effect in chronic hepatitis B Chan et al., 2003 P. urinaria extract Patients received P. urinaria extract lose detectable hepatitis B e-antigen from their serum and likely to seroconvert hepatitis B e-antibody status from negative to positive Wang et al., 1995 anti-URG11 and anti-URG19 levels were at relatively higher values of 68.0% (34/50), and 66.0% (33/50), respectively. Wang et al. (1995) report on 35 patients receiving a P. urinaria extract and thirty-five control patients; there was no detectable hepatitis B e-antigen in patient's serum after treatment with P. urinaria.
No patient changed status with respect to hepatitis B s-antigen . In contrast to the above results of anti-HBV effects of P. urinaria, an another study indicates that P. urinaria treatment for 6 months has no effect on HBV patients including no variation in log10 [HBV DNA] reduction using P. urinaria at 1 g (0.18 ± 1.42), 2 g (0.33 ± 1.08), or 3 g (0.85 ± 1.30) compared to a placebo (0.28 ± 0.85). Also there was no difference in the HBeAg conversion and ALT normalization of treated compared to control groups (Chan et al., 2003). Wong et al. (2013) using a tablet containing 400 mg of P. urinaria for 24 weeks find no improvement in non-alcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). Histologically, there is a minor reduction in steatosis and hepatocyte ballooning in the treated group, however, it is not significant. Perhaps, P. urinaria might not be a suitable agent to treat NASH (Wong et al., 2013).

Toxicology
Little data is available regarding the toxicity profiles of P. urinaria preparations. Chan et al. (2003) demonstrates that P. urinaria is well tolerated for 6 months by both male and female patients aged between 18 and 65 with positive hepatitis B surface antigen (HBsAg). There is no difference in toxicological measurements between treated and control groups; in both groups some subjects experienced mild negative effects.

FUTURE PROSPECTS
This review summarizes information regarding the traditional uses of phytochemicals, pharmacological activities of crude extracts as well as pure compounds, analysis of active compounds, and clinical trials related to P. urinaria. There is evidence that the crude extracts and pure compounds found within P. urinaria have anticancer, hepatoprotective, antimicrobial, antidiabetic, and cardioprotective activities through various signaling pathways. Although the chemical structure and its biological potential of some of the constituents are known, generally, the mechanisms of action need to be investigated for further development into therapeutics. Systematic efficacy studies are necessary to examine standardized extracts of P. urinaria and to identify the bioactive molecules responsible for the pharmacological activities. If possible, specific targets (i.e., receptors) need to be identified. The reported clinical data for P. urinaria against HBV is limited and consequently limits the use of herbal medicines to treat chronic liver disease. The compounds brevifolin and its derivatives, corilagin, ellagic acid, gallic acid, geraniin, loliolide, phyllanthin may be drug candidates for treating liver diseases because of their potent antiviral activites including antihepatitis activity. The high concentration of these compounds in P. urinaria suggests their use and indicates that studies are needed to assess the absorption, distribution, metabolism, and excretion of candidate compounds. Mechanism of action studies on the liver protecting effect of P. urinaria preparations and purified compounds when combined with conventional medicines, are also expected to lead the way in the discovery of new agents with improved pharmacological properties.
The herbal medicines cultivated in different geographical regions differ in their composition as well as their therapeutic effects demanding quality control of P. urinaria preparations and toxicological studies. Toxicological studies need to address the mycotoxin, heavy metal, and pesticide concentrations as well as the general toxicity of P. urinaria extracts and purified compounds. Attempts need to be made to gain regulatory approval of P. urinaria preparations as nutraceuticals or medicinal drugs.

AUTHOR CONTRIBUTIONS
MG wrote the manuscript. S-TD edited the manuscript. Both authors have seen and agreed on the finally submitted version of the manuscript.