Phyllanthin and related lignans were found to be responsible, at least in part, for most of the activity of Phyllanthus species. This observation encouraged the authors to develop methods for the preparation of an extract rich in phyllanthin and related lignans from the aerial parts of P. niruri L. Direct extraction with solvents produced extracts with variable yields and contents of lignans. Lignans were identified by LC-ESI-MS analysis as phyllanthin (used as marker substance), hypophyllanthin, phylltetralin, nirtetralin, and niranthin. Extraction with boiling water produced 18.10 g% (w/w) extract with a trace amount of lignans (phyllanthin content of 0.33 ± 0.10 mg/g extract), while extraction with MeOH gave 3.6 g% w/w extract with a low phyllanthin content (3.1 mg/g extract), as determined by HPLC. However, Soxhlet extraction with hexane, CH2Cl2, or acetone gave extracts with low yields (0.82, 1.12, and 3.40 g% w/w, respectively) and a higher phyllanthin contents (36.2 ± 2.6, 11.7 ± 1.68, and 11.7 ± 1.10 mg/g extract, respectively). Extraction quality and efficiency were optimized by adopting the following three different approaches: (1) Alkaline digestion of the plant material with 30% potassium hydroxide yielded 3.1 g% w/w of purified extract with high phyllanthin content (22.34 ± 0.13 mg/g); (2) microwave-assisted extraction using 80% MeOH gave an extract with a better yield (8.13 g% w/w) and phyllanthin content (21.2 ± 1.30 mg/g) (after filtration through a Diaion HP-20 column); and (3) treatment of the ground plant material at 50 °C with two hydrolytic enzymes, cellulase (9 U/g for 12 h) and then, protease (4 U/g up to 72 h) optimized the yield of extract (13.92 g% w/w) and phyllanthin content (25.9 mg/g extract and total lignans content of 85.87 mg/g extract). In conclusion, the nonconventional methods presented here are superior for optimizing the yield of extract and its lignan contents from the aerial parts of P. niruri.

The members of the genus Phyllanthus have long been used in the treatment of a broad spectrum of diseases. They exhibited antiproliferative activity against various human cancer cell lines. Breast cancer is the most diagnosed cancer and a leading cause of cancer death among women. Doxorubicin (DOX) is an anticancer agent used to treat breast cancer despite its significant cardiotoxicity along with resistance development. Therefore, this study was designed to assess the potential cytotoxicity of P. niruri extracts (and fractions) alone and in combination with DOX against naïve (MCF-7) and doxorubicin-resistant breast cancer cell lines (MCF-7ADR). The methylene chloride fraction (CH2Cl2) showed the most cytotoxic activity among all tested fractions. Interestingly, the CH2Cl2-fraction was more cytotoxic against MCF-7ADR than MCF-7 at 100 µg/mL. At sub-cytotoxic concentrations, this fraction enhanced the cytotoxic effect of DOX against the both cell lines under investigation (IC50 values of 0.054 µg/mL and 0.14 µg/mL vs. 0.2 µg/mL for DOX alone against MCF-7) and (1.2 µg/mL and 0.23 µg/mL vs. 9.9 µg/mL for DOX alone against MCF-7ADR), respectively. Further, TLC fractionation showed that B2 subfraction in equitoxic combination with DOX exerted a powerful synergism (IC50 values of 0.03 µg/mL vs. 9.9 µg/mL for DOX alone) within MCF-7ADR. Untargeted metabolite profiling of the crude methanolic extract (MeOH) and CH2Cl2 fraction exhibiting potential cytotoxicity was conducted using liquid chromatography diode array detector-quadrupole time-of-flight mass spectrometry (LC-DAD-QTOF). Further studies are needed to separate the active compounds from the CH2Cl2 fraction and elucidate their mechanism(s) of action.

Doxorubicin (DOX) is a cornerstone chemotherapeutic agent for the treatment of several malignancies such as breast cancer; however, its activity is ameliorated by the development of a resistant phenotype. Phyllanthus species have been studied previously for their potential anticancer properties. The current work is aimed to study the potential cytotoxicity and chemomodulatory effects of hypophyllanthin (PN4) and phyllanthin (PN5) isolated from Phyllanthus niruri to DOX against the adriamycin multidrug-resistant breast cancer cells (MCF-7ADR) and elucidate their mechanism of action. The major compounds of the active methylene chloride fraction were isolated and assessed for their potential cytotoxicity and chemomodulatory effects on DOX against naïve (MCF-7) and resistant breast (MCF-7ADR) cancer cells. The mechanism of action of both compounds in terms of their impacts on programmed/non-programmed cell death (apoptosis and autophagy/necrosis), cell cycle progression/arrest, and tumor cell migration/invasion was investigated. Both compounds PN4 and PN5 showed a moderate but similar potency against MCF-7 as well as MCF-7ADR and significantly synergized DOX-induced anticancer properties against MCF-7ADR. The chemomodulatory effect of both compounds to DOX was found to be via potentiating DOX-induced cell cycle interference and apoptosis induction. It was found that PN4 and PN5 blocked the apoptosis-escape autophagy pathway in MCF-7ADR. On the molecular level, both compounds interfered with SIRT1 expression and consequently suppressed Akt phosphorylation, and PN5 blocked apoptosis escape. Furthermore, PN4 and PN5 showed promising antimigratory and anti-invasive effects against MCF-7ADR, as confirmed by suppression of N-cadherin/β-catenin expression. In conclusion, for the first time, hypophyllanthin and phyllanthin isolated from P. niruri showed promising chemomodulatory effects to the DOX-induced chemotherapeutic activity against MCF-7ADR. Both compounds significantly synergized DOX-induced anticancer properties against MCF-7ADR. This enhanced activity was explained by further promoting DOX-induced apoptosis and suppressing the apoptosis-escape autophagy feature of the resistant breast cancer cells. Both compounds (hypophyllanthin and phyllanthin) interfered with the SIRT1/Akt pathway and suppressed the N-cadherin/β-catenin axis, confirming the observed antiproliferative, cytotoxic, and anti-invasive effects of hypophyllanthin and phyllanthin.