Characterisation of a New Fungal Immunomodulatory Protein from Tiger Milk mushroom, Lignosus rhinocerotis.

Lignosus rhinocerotis (Tiger milk mushroom) is an important folk medicine for indigenous peoples in Southeast Asia. We previously reported its de novo assembled 34.3 Mb genome encoding a repertoire of proteins including a putative bioactive fungal immunomodulatory protein. Here we report the cDNA of this new member (FIP-Lrh) with a homology range of 54-64% to FIPs from other mushroom species, the closest is with FIP-glu (LZ-8) (64%) from Ganoderma lucidum. The FIP-Lrh of 112 amino acids (12.59 kDa) has a relatively hydrophobic N-terminal. Its predicted 3-dimensional model has identical folding patterns to FIP-fve and contains a partially conserved and more positively charged carbohydrates binding pocket. Docking predictions of FIP-Lrh on 14 glycans commonly found on cellular surfaces showed the best binding energy of -3.98 kcal/mol to N-acetylgalactosamine and N-acetylglucosamine. Overexpression of a 14.9 kDa soluble 6xHisFIP-Lrh was achieved in pET-28a(+)/BL21 and the purified recombinant protein was sequence verified by LC-MS/MS (QTOF) analysis. The ability to haemagglutinate both mouse and human blood at concentration ≥0.34 µM, further demonstrated its lectin nature. In addition, the cytotoxic effect of 6xHisFIP-Lrh on MCF-7, HeLa and A549 cancer cell lines was detected at IC50 of 0.34 µM, 0.58 µM and 0.60 µM, respectively.

The Antiproliferative Activity of Sclerotia of Lignosus rhinocerus (Tiger Milk Mushroom).

Lignosus rhinocerus, the tiger milk mushroom, is one of the most important medicinal mushrooms used by the indigenous people of Southeast Asia and China. It has been used to treat breast cancer. A cold water extract (LR-CW) prepared from the sclerotia of L. rhinocerus cultivar was found to exhibit antiproliferative activity against human breast carcinoma (MCF-7) and human lung carcinoma (A549), with IC(50) of 96.7 µg/mL and 466.7 µg/mL, respectively. In comparison, LR-CW did not show significant cytotoxicity against the two corresponding human normal cells, 184B5 (human breast cell) and NL 20 (human lung cell). DNA fragmentation studies suggested that the cytotoxic action of LR-CW against cancer cells is mediated by apoptosis. Sephadex G-50 gel filtration fractionation of LR-CW yielded a high-molecular-weight and a low-molecular-weight fraction. The high-molecular-weight fraction contains mainly carbohydrate (68.7%) and small amount of protein (3.6%), whereas the low-molecular-weight fraction contains 31% carbohydrate and was devoid of protein. Only the high-molecular-weight fraction exhibited antiproliferative activity against cancer cells, with IC(50) of 70.0 µg/mL and 76.7 µg/mL, respectively. Thus, the cytotoxic action of the LR-CW is due to the high-molecular-weight fraction, either the proteins or protein-carbohydrate complex.

Protective effects of Mucuna pruriens seed extract pretreatment against cardiovascular and respiratory depressant effects of Calloselasma rhodostoma (Malayan pit viper) venom in rats.

The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.

The protective effects of Mucuna pruriens seed extract against histopathological changes induced by Malayan cobra (Naja sputatrix) venom in rats.

Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Nigeria as a prophylactic oral antisnake remedy. In the present studies, we investigated the protective effects of M. pruriens seed extract (MPE) against histopathological changes induced by intravenous injection of Naja sputatrix (Malayan cobra) venom in rats pretreated with the seed extract. Examination by light microscope revealed that the venom induced histopathological changes in heart and blood vessels in liver, but no effect on brain, lung, kidney and spleen. The induced changes were prevented by pretreatment of the rats with MPE. Our results suggest that MPE pretreatment protects rat heart and liver blood vessels against cobra venom-induced damages.

Natural biflavones as novel inhibitors of cathepsin B and K.

Cathepsin B and K, two important members in lysosomal proteases, involve in many serious human diseases, such as tumor and osteoporosis. In order to find their novel inhibitors, we performed the inhibition assays of cathepsin B and cathepsin K in vitro, randomly screened compounds from plants, and found six biflavones, named AMF1-5 and HIF, can potently inhibit cathepsin B and cathepsin K, especially AMF4 and HIF with IC(50) of 0.62 and 0.58 microM against cathepsin B. They are novel inhibitors for cathepsin B and K. Inhibition and flexible docking studies indicated that these biflavones are reversible inhibitors of cathepsin B, and their binding patterns and interaction modes with cathepsin B made them more specific to cathepsin B endopeptidase.