Studies On Germplasm Identification And Bioactivity Comparison Of Anti-Tumor TCM Mao-Ren-Shen And Its Related Species

Mao-Ren-Shen (cat ginseng) is one of the most frequently used traditional Chinese medicine (TCM) for anti-tumor therapy, especially against cancers of alimentary canal, in East China. It was recorded to be made from Actinidia valvata according to most TCM dictionaries and Flora. However, A. macrosperma, a morphologically similar species to A. valvata, has been believed to be the genuine resource of Mao-Ren-Shen and to be more effective than A. valvata in first and regularly applied area, based on our folk investigation. Enormous demands of this TCM led to over-exploitation of wild resource and confusion of original plant. Adulterants, such as A. melanandra, A. lanceolata, A. chinensis, were frequently observed to be misused as Mao-Ren-Shen resulted from wrong identification and profitable purpose. The quality and clinic effect were seriously jeopardized. The crucial issues are: i) What is the genuine resource of Mao-Ren-Shen; ii) how to identify it; iii) whether other Actinidia species can be used as it. To deal with these questions, we carried out a series of researches including original plant confirmation; morphological, chemical & physical, and molecular identification; inhibition activity on in vitro tumor cell line; analysis of mineral elements; analysis of essential oil. The results were obtained below.1. Confirmation of genuine resource of Mao-Ren-ShenA. macrosperma was proved to be the main resource of Mao-Ren-Shen, while the originally recorded resource A. valvata maybe the minor resouce at most, according to the results of folk and market survey, identification of plant and raw material, and history of medicinal application and scientific name.2. Germplasm identificationA. macrosperma was discriminated from adulterants comprehensively by methods of morphology of original plant and raw material, fluorescent reaction, UV spectrum, fluorescence spectrum, FT-IR, and PCR-RFLP. Raw material of A. macrosperma was completely and accurately identified by combination of morphology, fluorescent colors of 4 solvent extracts excited by 254nm and 365nm UV light, UV spectra of 4 solvent extracts, and fluorescent spectra of 2 solvent extracts. A discrimination key was presented finally. FT-IR spectra and subtraction spectra can distinguish raw materials of nine Actinidia species rapidly. Specific restriction patterns of trnK sequence from A. macrosperma digested by DdeI, DraI were obtained by PCR-RFLP. Molecular fingerprint of A. macrosperma was established.3. Comparison of inhibition activity on in vitro tumor cell linesThe results of tumor cell inhibition indicate A. macrosperma is effective to the studied three cell lines, but differ in intensity: SMMC-7221 (IC50=54.22μg/ml) > SGC-7901 (133.16μg/ml) > A549 (206.63μg/ml). In assessment of activity on liver cancer SMMC-7721, A. callosa var. discolor, A. hemsleyana, A. eriantha and A. lanceolata are more active than A. macrosperma, especially A. lanceolata with IC₅₀ of 6.53μg/ml, about 8 times that of A. macrosperma. With regard to stomach cancer SGC-7901 and lung cancer A549, A. macrosperma (IC50=133.16μg/ml, 206.63μg/ml) is as effective as A. valvata (136.46μg/ml, 224.83μg/ml), while the other species were ineffective. Therefore, our suggestion for application of Mao-Ren-Shen is that A. macrosperma and A. valvata can both be used as Mao-Ren-Shen for therapy of stomach and lung cancers, but A. valvata and other adulterants should not be used as Mao-Ren-Shen for liver cancer treatment. The high activity of A. lanceolata on liver cancer should be concerned.Based on the combination of HPLC profile and inhibition activity, it was speculated that the active components responsible for the cytotoxocity may be not the ones that the highest peaks in HPLC profile represent, while the ones unobviously detected. HPLC profiles need to be improved by modify elution methods.4. Analysis of mineral elementsMineral elements are deemed to benefit the effects of TCM, as well as bioactive fragment. Mineral elements of raw materials from nine Actinidia species were analyzed by ICP-AES. At least four mineral elements, Cu, Zn, Se, Fe, are believed to inhibit formation and growth of cancer. A. macrosperma is with the highest contents of Cu and Zn, relatively low content of Se, and middle content of Fe. It indicated that the effect of anti-tumor of A. macrosperma may be partly related to high contents of Cu and Zn. Cluster analysis was conducted based on mineral elements data with enlarged weights of Cu, Zn, Se, Fe. Cluster dendrograms showed A. macrosperma was quite different from the other eight species. Therefore, from the angle of mineral elements related to anti-tumor, it seemed other Actinidia species are not suitable to be used as Mao-Ren-Shen.5. Research on catnip responseMao-Ren-Shen first caught people's interests because of its attraction to cat. Cats preferred eating its fresh leaves and twigs to excite themselves and cure wounds. The reaction was named catnip response. Essential oils from fresh leaves of both wild and regenerated plants of A. macrosperma were analyzed by GC-MS. Three compounds, dihydronepetalactone, iridomyrmecin, and dihydroactinidiolide, were detected in essential oils from leaves of both wild plant and artificially cultured plant with similar contents. These compounds have been recognized to be responsible for catnip response.