Tu Jing Pi

Bioassay-directed fractionation identified pseudolaric acid B as the main antifungal constituent. It was active against Trichophyton mentagrophytes, Torulopsis petrophilum, Microsporum gypseum and Candida species, while its methylated or hydrolysed derivatives were not. Minimum inhibitory and fungicidal concentrations against Candida and Torulopsis were comparable with amphotericin B, and in a murine disseminated candidiasis model intravenous pseudolaric acid B significantly reduced recovered colony-forming units and prolonged survival. The plant is named here by the synonym Pseudolarix kaempferi.

Eight compounds were isolated from ethanol extracts of Cortex Pseudolaricis, the root bark of Pseudolarix amabilis, by bioassay-guided fractionation: pseudolaric acid A, ethyl pseudolaric acid B, pseudolaric acid B, pseudolaric acid B-O-beta-D-glucoside, piperonylic acid, propionic acid, 3-hydroxy-4-methoxybenzoic acid and 4-(3-formyl-5-methoxyphenyl) butanoic acid. All inhibited mycelial growth at 5 microgram/mL, with pseudolaric acids B and A strongest at EC50 1.07 and 1.62 microgram/mL. The pathogen is a plant fungus, so the relevance is to the potency ranking of the constituents rather than to human tinea.

Pseudolaric acid B inhibited fluconazole-resistant and fluconazole-susceptible Candida tropicalis alike, with median MICs of 8 to 16 microgram/mL. Combined with fluconazole it was synergistic in all 13 resistant strains and in 2 of 9 susceptible strains, with fractional inhibitory concentration indices from 0.070 to 0.375, and the combination removed more than 80% of early and mature biofilm.

In a rat model of fungal keratitis, pseudolaric acid B destroyed hyphal integrity, lowered clinical scores and fungal burden and reduced inflammatory cell infiltration, acting through SIRT1 to modulate Nrf2 and NF-kappaB signalling. No toxicity was seen in vivo or in vitro below 0.3 microM, which also indicates how narrow the tested safe range is.

Direct biochemical characterisation showing that pseudolaric acid B binds at the colchicine site of tubulin. This is the molecular basis for its microtubule-destabilising, antimitotic and antiangiogenic activity and for the mechanistic caution about co-exposure to other tubulin poisons.

Neither pseudolaric acid A nor B showed oestrogenic or antioestrogenic activity or inhibited decidual formation. After an effective dose of pseudolaric acid B at 30 mg/kg on day 6 of pregnancy in rats, plasma progesterone, oestradiol and prostaglandins E and F and uterine prostaglandins were not significantly reduced. Both acids at 200 microgram/mL damaged only part of the decidual and trophoblast cells of human uterus in vitro and reduced contractile tension in isolated early-pregnant rat uterine muscle. A low dose of 2 mg/kg/day on days 6 to 12 still lowered fetal body weight and length and placental weight. The authors attribute the abortifacient effect to ischaemia from a vasoconstrictor action.

Endometrial and myometrial blood flows were measured by hydrogen gas clearance in 336 anaesthetised rats given pseudolaric acid B by gavage at 20 or 30 mg/kg on days 7 to 9 of pregnancy. Endometrial flow fell by 33 to 66% and myometrial flow by 30 to 65% against vehicle, uterine weight stopped increasing on days 10 to 14, and systemic arterial pressure was relatively unchanged. The authors conclude the local fall in uterine blood flow is the most important cause of embryonic death.

Pseudolaric acid B injected into the hamster ovarian bursa before ovulation significantly reduced the fertilisation rate of ova, with no effect on spermatozoal activity or fertilising ability. Above 50 microgram/mL in medium it markedly reduced fertilisation of ova with or without cumulus; at 5 microgram/mL only cumulus-free ova were affected. Oral dosing at 20 mg/kg daily for 4 days before mating produced a partial antifertility effect.

Reviews pseudolaric acid B as a microtubule-destabilising agent that disrupts mitotic spindles and causes G2/M arrest, induces apoptosis through Bcl-2/Bax, caspase-dependent and caspase-independent routes, reactive oxygen species and MAPK signalling, inhibits angiogenesis by promoting proteasomal degradation of VEGF and HIF-1alpha, and reverses multidrug resistance via Cox-2, PKC-alpha and P-glycoprotein. It is a preclinical review; it reports no antifertility, hepatotoxicity or nephrotoxicity data, which sit in the older Chinese pharmacology literature.

HPLC quantification across five plant parts found pseudolaric acid B at 3.39 mg/g in root bark, 0.273 mg/g in the inner root, 0.215 mg/g in stem bark, 0.076 mg/g in leaf and 0.041 mg/g in cone. Root bark is roughly 12 times richer than stem bark and 45 times richer than leaf, which is the pharmacognostic reason the drug is specified as root bark or near-root trunk bark rather than any convenient bark.

(+)-Catechin from Pseudolarix amabilis was active against the monogenean fluke Dactylogyrus intermedius in goldfish. It is supporting evidence that the parasite-killing reputation of the drug is not carried by the pseudolaric acids alone, but the model is a fish ectoparasite and does not transfer to human parasitology.