2018/05/30_FN

Question and Answer

th
65 Japanese Biochemical Society Meeting Kinki Branch

1. Q: Did you check the efficacy of the herb in molecular level (DNA or RNA)?
A: Not yet, but I will do in the future after isolate constituent

2. Q: Is there any other way to check anti-inflammatory effect of your extract?

A: Yes, there are. We can check the expression of pro-inflammatory cytokines such as
IL-6 and TNF also their transcription factor (NFk).

3. Q: There are another type of NOS protein, do you plan to analyse the expression of
another NOS protein? I think you should check another too.
A: Not decided yet. Because in this research we use model of cytokines-signalling (IL-
1) to mediate nitric oxide production which is produced by iNOS. So, we only analyse
iNOS expression.

4. Q: Based on the decreasing of iNOS protein expression, seems not really significant
different, is it related to gene, protein or mRNA stability or another factor?
A: I am not sure but I think not. Also, we do not not check in RNA level so still cannot
judge the reason.

5. Q: What kind of disease that can be cured by this herb?

A: this herb reported to cure cardiovascular disease, neuroinflammation, and also this
herb contained in kampo drug, Yokukansan, that used to treat dementia and Alzheimer.

6. Q: Is this herb has efficacy to another disease besides inflammatory disease?

A: Yes, some paper reported this herb as anticancer and antifungal.

7. Q: How do you obtain the cells? Why do you use primary rat hepatocyte instead of
cell lines?
A: the primary cells was obtained by isolate from rat liver. We use rat primary cell
culture instead of cell lines due to their characteristic. Primary cell culture more
representative model for evaluate NO production, since they still have natural features
related to response to cytokine. Cell lines probably loss their natural features during their
tumorigenesis.

8. Q: do you plan to use another model for evaluate the herb efficacy, such using
Drosophilla or bacteria?
A: If another simpler organism, we are not planning to do that. But we plan to do in vivo
experiment (animal model such as mice). Because this research performed basically to
discover drug candidate, so we want to use model with similar condition as this drug will
be administrated, absorbed in the body and regulate protein or gene that responsible for
related disease.

9. Q: Why do you use methanol instead of ethanol, since methanol has known more
toxic than ethanol?
 A: We use methanol because we want to obtaine more extract yield. Due to toxicity, after
we did NO measurement, we also did Lactate dehydrogenase measurement to know
whether the extract is cytotoxic or not. Since the LDH level is low, it means that there is
no problem with methanol as solvent for extraction.

10. Q: How do you dilute the extract before applied to medium culture?
A: I use DMSO to dilute extract.

11. Q: How much DMSO percentage did you use in culture medium? We knows that
DMSO is cytotoxic too.
A: I use less than 1% DMSO in culture medium. Based on literature, it is safe percentage
in culture medium.

12. Q: Will you do GC, HPLC, and another chemistry analysis to identify the
compounds?
A: Yes, but still in progress.