The Genus Phyllanthus: An Ethnopharmacological, Phytochemical, and Pharmacological Review

The plants of the genus Phyllanthus (Euphorbiaceae) have been used as traditional medicinal materials for a long time in China, India, Brazil, and the Southeast Asian countries. They can be used for the treatment of digestive disease, jaundice, and renal calculus. This review discusses the ethnopharmacological, phytochemical, and pharmacological studies of Phyllanthus over the past few decades. More than 510 compounds have been isolated, the majority of which are lignins, triterpenoids, flavonoids, and tannins. The researches of their remarkable antiviral, antioxidant, antidiabetic, and anticancer activities have become hot topics. More pharmacological screenings and phytochemical investigations are required to support the traditional uses and develop leading compounds.


Introduction
Phyllanthus (Euphorbiaceae) is a large genus and widely distributed in tropical and subtropical zones like tropical Africa, tropical America, Asia, and Oceania. This genus, consisting of more than 700 species, can be classified into 11 subgenuses [1,2]. The most popular 24 species are chiefly belonging to subgenus Kirganelia, Cicca, and Phyllanthus and they are traditionally used by different nationalities.
Genus Phyllanthus has been employed as herbal drugs for a long time in China, India, Brazil, and Southeast Asian countries. The most abundant species are used in India and have a beneficial role in Ayurveda for the treatment of digestive, genitourinary, respiratory, and skin diseases [3,4]. In China, herbs and their prescriptions are used to treat hepatitis B, hypertension, dropsy, and sore throat [2]. These herbal drugs are employed by local inhabitants of Thailand, Latin America (especially Brazil), and Africa to cure jaundice, renal calculus, and malaria, respectively [5][6][7].
By virtue of the wide uses of Phyllanthus as anti-HIV, anticancer, and anti-HBV agents, there has been considerable interest in the investigations of this genus in recent years and the researches about pharmacology and chemistry had been finished in a deep going way. This report reviews the ethnopharmacological, phytochemical, and pharmacological investigations of Phyllanthus over the past few decades. More than three hundred articles were selected from the data taken from SciFinder Scholar database by searching the keyword "Phyllanthus".

Tannins.
Tannins were progressively reported from the genus Phyllanthus since 1992. Hydrolyzable tannins  are characterized by the presence of one or more galloyl, hexahydroxydiphenoyl (HHDP), and HHDP metabolites attached to a glucopyranose core, which are mainly isolated from P. emblica, P. amarus, P. niruri, and P. urinaria. Compounds 271-279 are condensed tannins, which are the condensation of flavan-3-ols and linked by C-C. A great many condensed tannins were proved to have antiviral activity [53].
Ellagitannins  are the largest group of hydrolyzable tannins. Corilagin and geraniin are most extensively obtained from this genus and are characteristic compounds of ellagitannins, which exhibited multiple activities such as antioxidant, anti-HIV, antitumor, and antihyperalgesic activities [6,111,188,195,196,199,201,202].  [151,171,178,195,203,224].

Sterols.
Until now, thirty sterols (334-363) from Phyllanthus have been reported. All the sterols are phytosterols with a side chain (C8-C10) substitution at C-17, and half of which were isolated from P. emblica.

Phenols and Others.
Compounds 396-468 belong to phenols, which have one and several phenolic hydroxyl groups. Thirty other constitutions (469-512) have been isolated. Mucic acid (compounds 445-455) and its derivatives (compounds 498-499) can only be found in P. emblica among this genus.

Biological Activity
The remarkable traditional uses of genus Phyllanthus lead to the various researches of biological activities, such as antiviral, antioxidant, antidiabetic, anticancer, and immunomodulatory activities. In this section, biological activity researches of the extracts of the plants are highlighted.

Antiviral Activity.
Various Phyllanthus plants were reported to have strong antiviral potential such as anti-HIV, anti-HCV, anti-HSV, and anti-HCMV. The aqueous extract of P. emblica reduced viral load of HIV significantly at the dose of 400 g/mL [282]. DNA-polymerase and ribonuclease H (RNase H) activities of HIV-1 reverse transcriptase were inhibited by aqueous extract of P. sellowianus with IC 50 values of 2.4 ± 0.8 g/mL and 5.9 ± 1.4 g/mL, respectively [283]. Moreover, methanol extract of P. reticulatus strongly Evidence-Based Complementary and Alternative Medicine 23 inhibited the activity of RNase H by 99% at the dose of 50 g/mL [284]. HCV-infected HuH7 cells were used to test the anti-HCV activities of methanolic fraction of P. amarus. The fraction was proved to suppress the replication of HCV monocistronic replicon RNA and HCV H77S viral RNA without toxic effect in host cells. Inhibiting HCV-NS3 protease enzyme and NS5B enzyme may be the main mechanism [285]. Aqueous extract of P. orbicularis revealed inhibition activity against the replication of HCMV, HSV-1, and HSV-2 as well as BHV-1 with EC 50 values of 57.7, 28.8, 25.7, and 21.27 g/mL, respectively. The selectivity indexes (SI) were ranged from 8.7 to 37.6 [286,287].
Friend murine leukemia virus (FMuLv) induced erythroleukemia in BALB/c mice was relieved by metabolic extract of P. amarus. The extract inhibited leukemic cells from infiltrating into the sinusoidal space, decreased the morbidity of anemia, and improved survival rate of leukemia animals. Besides, the extract induced the upregulation of p53 and p45NFE2 and downregulation of Bcl-2 in the spleen [288].

Antioxidant Activity.
Methanolic and aqueous parts of this genus have remarkable antioxidant activity, which may be correlated with the hydroxyl rich compositions. P. acidus, P. polyphyllus, and P. fraternus showed remarkable hepatoprotective activity against liver toxicity which was induced by acetaminophen, carbon tetrachloride, bromobenzene, and thioacetamide [42,[289][290][291]. The biochemical parameters as well as antioxidants levels were restored by these parts at the dose of 300 mg/kg. What is more, mitochondrial dysfunction in liver, induced by bromobenzene, was relieved by prior oral administration of aqueous part of P. fraternus at the dose of 100 mg/kg [51,291].
Antimycin A governed mitochondrial protein degeneration, lipid peroxidation and mitochondrial DNA damage, and H 2 O 2 induced membrane damage of Hep3B cells were considerably mitigated by aqueous fraction of P. amarus [164]. Mutagenesis induced by PhIP and 4-ABP and DNA damage induced by -ray and UVB were protected by aqueous fraction of P. orbicularis [292][293][294].
Methanol extract of P. debilis showed strong antioxidant activity when tested by various antioxidant assays including total antioxidant, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and nitric oxide scavenging assays. Besides, further study demonstrated that total phenolic was correlated with antioxidant activity [52]. In addition, hydromethanolic extract of P. virgatus exhibited substantially antioxidant capacity in both DPPH scavenging (IC 50 = 30.4 g/mL) and linoleic acid oxidation inhibiting (84%) method [5].
After oral aqueous extract of P. niruri for 28 days, the levels of LPO and MDA were decreased while the concentrations of SOD, CAT, and GPx were increased. After being pretreated with the aqueous fraction of P. sellowianus, hemorheological parameters were ameliorated and red blood cells (RBCs) showed large globular aggregates and agglutination [301].
Ehrlich ascites carcinoma tumor model was used to evaluate the antitumor activity of P. polyphyllus. Oral administration of methanol fraction at the dose of 200 mg/kg could significantly reduce the solid tumor volume. Hematological parameters, protein, packed cellular volume (PCV), and antioxidant enzymes such as LPO, GPx, GST, SOD, and CAT were greatly regulated [57].

Immunomodulatory
Activity. Ethanol extracts of P. urinaria and P. amarus were demonstrated to have inhibitory effects on the chemotaxis of neutrophils and monocytes with IC 50 lower than 2.92 g/mL. In addition, phagocytic activity and CD18 expression of neutrophils and monocytes were downregulated [163].
Oral administration of P. reticulatus extract at the dose of 100 mg/kg demonstrated a significant increase in phagocytic activity, the percentage of neutrophil adhesion, and white blood cell in albino mice [310].
4.6. Analgesic Activity. The extracts of P. corcovadensis, P. niruri, and P. tenellus showed significant reduction in writhing response induced by acetic acid, with ID 50 values of 30,19, and >30 mg/kg, respectively. The late phase of formalininduced pain could be relieved by P. tenellus with ID 50 of 100 mg/kg and both phases of formalin-induced pain could 24 Evidence-Based Complementary and Alternative Medicine be reduced by P. corcovadensis and P. niruri with ID 50 values of 100 and 52 mg/kg, respectively. The analgesic effects could not be antagonized by naloxone [311]. In addition, intraperitoneally given hydroalcoholic extracts of P. amarus, P. orbicularis, and P. fraternus produced a marked analgesic activity by inhibiting acetic acid-induced abdominal constriction, capsaicin-induced neurogenic pain, and late phase of formalin-induced paw licking [312]. The ethanol and aqueous extracts of P. emblica succeeded in inhibiting acetic acid-induced writhing response but failed in the tailimmersion test [313].

4.7.
Anti-Inflammatory Activity. In recent years, different inflammatory models such as Freund's complete adjuvant induced arthritis, carrageenin induced paw edema, and cotton pellet induced granuloma were employed to evaluate the anti-inflammatory effect of Phyllanthus. After receiving the aqueous extract of P. amarus, indexes of arthritis, joint diameter, and paw volume were decreased and thresholds of mechanical hyperalgesia and nociceptive were increased [314]. The ethanol fraction of P. simplex ameliorated the parameters of paw edema and granuloma and substantially inhibited nitric oxide (NO) production [315].

Antispasmodic
Activity. Isolated rabbit jejunum and guinea-pig ileum were employed for the in vitro tests for the antispasmodic effects of P. emblica. Carbachol and K + induced contractions of rabbit jejunum were released by the extract with IC 50 values of 0.09 mg/mL and 1.38 mg/mL. The pretreatment of guinea-pig ileum with the extract at 0.3 mg/mL caused a rightward parallel shift in the concentration-response curves of acetylcholine without suppression of the maximum contractile response. Dual blockade of muscarinic receptors and Ca 2+ channels can explain its antispasmodic activity [316].

4.9.
Hypotensive and Hypolipidemic Activity. Aqueous extract of the leaves of P. amarus was found to restrain both force and rate of myocardial contraction and to inhibit the intrinsic myogenic contraction of isolated rat portal vein [317]. Aqueous part of P. reticulatus was effective in releasing total cholesterol, lipid profile, and oxidative stress in hypercholesterolemic albino rats after oral administrated for 45 days at 250 mg/kg [14].

Wound
Healing. Extracts of P. emblica and P. niruri were demonstrated to have wound healing effect. Topical application with P. emblica could promote the proliferation of cells and cross-link of collagen in the full thickness excision wound [318]. Oral administration of P. emblica at the dose of 60 mg/kg showed healing effect against NSAID-induced gastric ulcer through upregulating the concentration of IL-10 and downregulating the levels of TNF-and IL-1 [319]. After treatment with P. niruri at the dose of 200 mg/kg, 98.8% of wound could be recovered in the excision and incision wound models on the 16th day [320].

Antimalarial Activity.
Malaria is a prevalent disease in many tropical and subtropical countries and folks of these places especially African people employed Phyllanthus as antimalarial agency. Plasmodium falciparum was suppressed by ethyl acetate fraction of P. acidus with IC 50 of 9.37 g/mL, and the SI equals 4.88 for HEp-2 cells and 11.75 for Vero cells [321]. What is more, chloroquine-resistant P. falciparum could be exhibited by P. amarus and P. muellerianus with IC 50 values of 11.7 and 9.4 g/mL, respectively. P. amarus presented protection effect on human RBCs damage caused by the virus [322]. The SI of P. muellerianus was higher than 5.3 for L-6 and MRC-5 cell lines [25,202].

Antidepressant
Activity. The aqueous extract of P. emblica (200 mg/kg) significantly decreased immobility period in both tail suspension test and forced swim test by decreasing the levels of MAO-A and GABA [323]. In the plus-maze, Hebb-Williams maze, and passive avoidance apparatus test, preparation of P. emblica produced a dosedependent upgrade in scores. The preparation was also proved to reverse the amnesia induced by diazepam and scopolamine and to reduce the cholinesterase activity and total cholesterol level in brain [324,325].
Aqueous extract of P. acidus was proved to regulate electrolyte transport in cystic fibrosis airways by increasing the intracellular levels of cAMP and Ca 2+ , stimulating basolateral K + channels, and activating and redistributing cellular localization of cystic fibrosis transmembrane conductance regulator [328].
Eight hours after being treated with the aqueous extract of P. sellowianus at a dose of 400 mg/kg, urine output of test animals was decreased from 2.59 to 3.69 mL/100 g [329].

Clinical Studies
The extracts of P. niruri were proved to have immunomodulatory effect and played a crucial role in treating pulmonary tuberculosis and vaginal candidiasis as well as varicella. In patients with pulmonary tuberculosis, after oral administration of P. niruri 50 mg/mL for 2-6 months, the level of IL-10 was decreased and the levels of plasma IFN-and TNF-were significantly increased. After 1-month treatment, the increase of the ratio of CD4 + /CD8 + was observed. In the vaginal candidiasis patients, after receiving P. niruri 100 mg/mL for 1-3 months, the levels of IFN-and IL-12 were elevated. As for varicella patients, the number of papules and the number crusts were decreased after treatment with the extract at the dose of 5 mg/mL [330].
Clinical studies of P. niruri in Brazil had been finished, from which the P. niruri showed beneficial effects on the treatment of urolithiasis. After 3-month treatment, calculi elimination was increased. Furthermore, urinary calcium excretion and residual stone fragments after lithotripsy were decreased. Toxic effects on kidney, cardiovascular, and nervous systems were not found [331].
In China, the clinical study of P. urinaria in treating chronic hepatitis B with 140 patients was well established. The results indicated that, after treatment with P. urinaria capsule for 3 months or 2 years, especially in the long term, the recovery rate in the index of HBV-DNA and HBeAg was 88.2% and 52.5%, respectively. Once the treatment stopped, the recurrence rate was 10.4% to 13.4% [332].

Toxicity Studies
After given aqueous leaf extract of P. niruri at the dose of 2000 mg/mL, no acute toxicity was observed at the levels of bilirubin, ALT, AST, total protein, albumin, globulin, ALP, GGT, urea, creatinine, full blood count, and hemoglobin [333]. After being treated with ethanol extract of P. niruri over a period of 90 days at doses of 30 and 300 mg/kg, the rats showed no genotoxic effect at the test of PCE/NCE ratio [334]. Reproductive toxicity of P. niruri was tested using estrogen values, progesterone values, and testosterone levels. The estrogen and progesterone levels increased more than 1.5fold above the control group after receiving 50 and 500 mg/kg aqueous leaf extract for 90 days, which reminded us of the cytotoxic of male antifertility properties [335].
Nephrotoxicity including interstitial oedema and tubular necrosis were detected after receiving 400 and 800 mg/kg of aqueous extract from P. amarus for 30 days [336]. The test animals were given 800 and 1600 mg/kg of the aqueous extract of P. amarus for 10 days, and significant pathological changes were found in the liver, kidney, and testis. The frequency of MNPCE, sperm abnormalities, total white blood cell, and lymphocyte counts were significantly increased, which suggested the genetic and systemic toxicity of P. amarus [337]. In addition, aqueous, methanolic, and hydromethanolic extracts of P. amarus (400 mg/kg) reduced locomotor activity and showed CNS depressant effect [338].
The LD 50 of ethanolic extract from P. fraternus was 1125 mg/kg in the toxicity test. When the rats received the extract at doses of 400 mg/kg for 7 days, no toxicity was detected in liver and kidney [339]. Hydroethanolic extract P. fraternus showed the quick onset and long duration of reduction of locomotor activity at the dose of 400 mg/kg [338].

Conclusion
514 compounds have been isolated from different species of Phyllanthus, including 126 terpenoids, 102 phenylpropanoids, 73 phenols, 54 flavonoids, 53 tannins, 33 sterols, 31 alkaloids, and a number of other compositions. Their wide range of biological activities such as antiviral, antioxidant, antidiabetic, anticancer, anti-inflammatory, hypolipidemic, immunomodulatory, and antidepressant activities are tested using polar solvents (water, methanol, and ethanol) extracts. These extracts are considered rich in phenols, flavonoids, and tannins, which may exhibit antioxidant activity in different degree due to their hydroxyl [340]. Consequently, most bioactivities of Phyllanthus may be correlated with the hydroxyl rich compounds.
In recent years, the traditional uses of Phyllanthus had been partly confirmed, and more evidences such as pharmacological researches and clinical studies are urgently needed to be taken. Further studies of phytochemical discovery and subsequent screenings are necessary to be taken to extend the use of Phyllanthus and to develop leading compound.