Comparison of the Biological Activity and Constituents in Japanese Ambers

Background/Aim: Kuji amber is an interesting natural source for drug dis-covery because a new anti-allergic compound, named kujigamberol and several new compounds have been isolatated from it. It was important to evaluate the yield, biological activities and constituents of each methanol extract of Kuji, Iwaki, Choshi, Mizunami and Ube ambers in Japan in order to establish if additional new compounds could be identified in these ambers. Materials and Method: Biological activities of each extract were evaluated using growth-restoring activity of the mutant yeast strain involving Ca 2+ -signal transduction and inhibition activity of degranulation in rat basophilic leukemia (RBL)-2H3 cells. Constituents of each extract were analyzed by high performance liquid chromatography (HPLC). Results: All ambers except Ube amber have growth-restoring activity against the mutant yeast. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by the calcium ionophore A23187 in a dose dependent manner. The main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Conclusion: Kuji and Iwaki ambers appeared to have the same origin. Choshi, Mizunami, and Ube ambers are valuable sources for biologically active compounds which are different from those of Kuji amber.


Introduction
Amber is polymerized and fossilized tree resin which is found worldwide and is *Authors with equal contributions.
A main new compound from Kuji amber, named kujigamberol, has potent anti-allergic activity against a rhinitis model through the inhibition of Ca 2+ -influx and leukotriene C4 production in rat basophilic leukemia (RBL)-2H3 cells [12].
Thus, ambers from Japan such as Kuji amber are not only fascinating natural sources for the study of organic geochemistry, but also for drug screening. A question that arises is why we can isolate new compounds from Kuji amber but isolate only known compounds from Baltic, Dominican and Burmese ambers.
As the environment of the earth on the Cretaceous-Paleogene (K-Pg) (formerly Cretaceous-Tertiary, K-T) boundary (65 Ma) changed to destroy most of plants and dinosaurs, the environment for the formation of Kuji amber that is older than the K-Pg boundary may be crucial for isolating new compounds. In contrast, both Baltic and Dominican ambers are younger than the K-Pg boundary.
Additionally, no new compound, such as kujigamberol, has been isolated and detected from Burmese amber that is older than Kuji amber [10]. Recently it was reported that kujigamberol was not detected in the Upper Apitiam-Lower Albian amber deposit of the Peñacerrada II (Basque-Cantabrian Basin, Spain) [13]. In this study, we examined the biological activities and their constituents of Iwaki, Choshi, Mizunami and Ube ambers and compared them to those from Kuji amber.

Materials and Methods
General experimental procedure: Kuji amber was excavated from mines of Kuji Kohaku Co. Ltd. located in the upper part of the Tamagawa Formation of Kuji Group of Kuji city, Iwate Prefecture. The other Japanese ambers were excavated in small quantity from each area ( Figure S1(a)). The yeast strain is a de-rivative of strain W303-1A and is YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1::hisG URA3 hisG pdr3::hisG) [3] [4]. Difco  YPD broth and YPD agar were obtained from Becton Dickinson (Franklin Lakes, NJ, U.S.A.). FK506 (tacrolimus) was kindly provided by the Fujisawa Pharmaceutical Co., Ltd. (now Astellas Pharma Inc., Tokyo, Japan). Unless otherwise stated, chemicals used were of the best commercially available grade.
Biological activities of each ambers against the mutant yeast: All ambers were ground to a powder and extracted with methanol (MeOH) for three days at 23˚C. After evaporation of the MeOH, each dried extract was weighed. Extracted samples of 10 mg/ml in MeOH were prepared for subsequent assays. Screening was carried out using the YNS17 strain and 5 μl of each sample was added to a plate as described previously [4]. The inhibitory activity of the Ca 2+ -signal transduction was determined by way of the strength and/or distinction of the yeast growth zone. An immunosuppressive drug, FK506 (2.5 ng/spot) was used as a positive control.

Results and Discussion
The weight of the MeOH extract of each amber: The yield of the MeOH extract of Kuji, Iwaki, Choshi, Mizunami and Ube was 3.4%, 4.4%, 1.1%, 26.7% and 33.8%, respectively. The yield of the MeOH extract from their domestic ambers is roughly proportional to each reported age [ Figure S1 Figure   S7(b)] was also isolated and identified from Iwaki amber and this suggested that it is from the same origin as Kuji amber.

Conclusion
The yield of methanol soluble fraction decreased with the age of amber. All ambers except Ube amber have growth-restoring activity against the mutant yeast strain involving Ca 2+ -signal transduction. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by A23187 in a dose dependent manner. Kuji and Iwaki ambers appeared to have the same origin, because amberene and 1-methlamberene were identified in both by HPLC. Additionally, the main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by MS and NMR. Choshi, Mizunami, and Ube ambers are therefore valuable sources for biologically active compounds which are different from those of Kuji amber. Advances in Biological Chemistry