Cold Vacuum Extracts of Double Cherry Blossom (Gosen-Sakura) Leaves Show Antitumor Activity

The present research examines the possibility of finding bio-molecular compounds from the double cherry blossom (termed as ‘Gosen-Sakura’ of Gosen-city, Niigata-prefecture, Japan) leaves, which have been long used in the preparation of the traditional Japanese sweet (wagashi) – ‘sakura-mochi’. Based on its indicated anti-microbial properties historically, our study provides a new low temperature vacuum extraction method for extracting ‘near natural form of water soluble leaf (cell) extracts from the Gosen-Sakura, and demonstrates the presence of some ‘novel’ compound(s) with anti-tumor cell lines proliferation inhibitory affects through the MTT assay. To our knowledge, no reports exist on the sakura tree ‘leaf (cell) extracts’ inhibiting tumor cell line growth. We further examined and compared the effects of known compounds with anti-tumor activity, coumarin and benzyl alcohol with Gosen-Sakura leaf extract; results lead us to hypothesize that the Gosen-Sakura leaf extract contains substance(s) other than the above 2 known compounds, with antitumor effect. Additionally, we speculate on the underlying mechanism of action of the Gosen-Sakura leaf extract by targeting cell division at the point of DNA synthesis and causing apoptosis. In conclusion, we present scientific evidence on the presence of a certain ‘novel’ biomolecule(s), with anti-tumor activity, in the Gosen-Sakura leaf which has been long used as a Japanese – the ‘sakura-mochi’.


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Sakura (cherry blossom) is a generic term for deciduous trees of the family 60 Rosaceae (sub-family, Amygdaloideae; Tribus, Amygdaleae; Genus, Prunus; 61 Sub-genus, P. subg. Cerasus; Sectio, P. sect. Cerasus; Nothospecies, Prunus × 62 yedoensis; Cultivar, P. y. 'Jindai-akebono -P. y. 'Somei-yoshino') represented by 63 Prunus cerasus or Prunus yedoensis [1][2][3]; and is very familiar to the Japanese with a 64 long cultural history. Cherry blossoms are popular as landscapes and are planted 65 everywhere in Japan appreciated not only for their natural beauty (sakura viewing) but 66 also in food as ingredients in tea and Japanese sweets mostly for its fragrance following 67 the salt fermentation of the petals. Other than the petals, the leaves of the sakura tree are 68 also used in the preparation of Japanese sweets since ancient times, and famously called 69 the `sakura-mochi` [4]; the sweet is eaten all over Japan during the sakura season. The 70 sakura-mochi is prepared from pounded glutenous rice which contains sweet red bean 71 paste wrapped around by a `salted` sakura leaf of usually the double cherry blossom [4]. 72 Though there are no direct evidences to the said anti-microbial and anti-bacterial 73 properties of the sakura-leaves, few researches on the antioxidant activity of Prunus 74 species are available [5][6][7][8]. 75 The reason behind the use of the leaves of the sakura tree, in particular the 76 double cherry blossom leaves, for edible use is thought to lie in its numerous relaxing effects. Since ancient times, the boiled cherry leaves extracts liquid has been 82 used for preventing skin roughness [11], has anti-inflammatory action for the skin [12], 83 and suppression of melanin production [13,14]. 84 Although the double cherry blossom is present all over Japan, we Table 1. In the case of adherent cells (Table 1), 96-well culture plates (Corning Inc.

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New York, USA) were inoculated with 3 × 10 4 cells / 100 μL / well of each cell and 165 pre-cultured for 24 hours at 37 °C in 5% CO 2 . After pre-incubation, the culture was 166 exchanged for a test medium and incubated at 37°C under 5% CO 2 for 24 to 48 hours.

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To the test medium, a Gosen-Sakura leaf extract was added to a final concentration of In the case of non-adherent (floating) cells (Table 1), the culture adjusted to 172 have 3 × 10 6 cells / 10 mL and was pre-cultured for 24 hours at 37°C in 5% CO 2 in a  are not used at all, it is possible to extract aromatic substances derived from the 100% 280 raw materials, and, iii) the technology is based on an applied machine being 281 commercialized as a dryer, and it is relatively inexpensive and easy to operate.

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In the case of the Gosen-Sakura, following the low temperature vacuum 283 extraction process, we obtained the leaf extract (around 95%) and cell dry powder (5%).

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These were used for downstream experiments as described below. combining benzyl alcohol with coumarin, a growth inhibitory effect was observed.

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However, since the cell viability did not decrease below 60% or less even under the 327 added condition of 5% (benzyl alcohol with coumarin), it was suggested that the 328 Gosen-Sakura leaf extract contains a substance having antitumor effect in addition to 329 the main components coumarin and benzyl alcohol (Fig 4). repaired. Therein, the rationale for the next experiment as performed below.

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Gosen-Sakura leaf extract induces apoptosis 356 Results of the HeLa (Fig 6) and A549 (Fig 7) tumor cells by APOPercentage The novelty of the study is once again highlighted in the fact that the Gosen-Sakura 382 leaves extraction was carried out using low temperature and where the method was  Table 1.    Table 1. The Cell Lines.