In order to develop functional (immune) foods, the objective is to compare the content of 2,6-dimethoxy-1,4-benzoquinone
(DMBQ) in different extracts prepared from barley germ
(solvent extraction - chloroform/ethyl acetate, primary and
secondary supercritical extraction, primary and secondary
supercritical encapsulation extraction).

By analyzing the batch extraction yield and DMBQ content of
two raw materials prepared during supercritical extraction
processes (solvent partition extraction and supercritical
encapsulation extraction), it was found that the supercritical
solvent partition extraction exhibited excellent turbidity and
viscosity, but with lower DMBQ content. On the other hand,
the supercritical encapsulation extraction had lower extraction
yield but relatively higher DMBQ content. Therefore, depending
on the intended use in the future, these two raw materials
can be selectively applied for commercialization.

To conduct a cell toxicity test of wheat germ extract using
RAW264.7 (macrophage) cells. RAW264.7 cells are used as
macrophages in in vitro experiments. Macrophages are
involved in the innate immune response, and the experiment
evaluates the immunomodulatory activity of substances by
measuring the secretion of nitric oxide (NO) and cytokines
produced after stimulation with LPS (lipopolysaccharide)
and other stimuli.

There were no significant differences in toxicity observed
between wheat germ powder extract and supercritical extract
at their respective concentrations.
Additionally, no significant toxicity differences were observed
between the extracts.

To validate the efficacy of wheat germ extract as an
immune-enhancing functional ingredient (Nitric oxide is
produced by the action of the iNOS enzyme in macrophages,
and its absorbance is detected using Griess reagent).

Compared to red ginseng extract, wheat germ extract
demonstrated a significant increase in nitric oxide production,
which was directly proportional to the concentration.
Moreover, at certain concentrations, its results were similar to
those of red ginseng.
This outcome yielded significant results for the nitric oxide
immunological assay indicators.

To validate the efficacy of wheat germ extract as an
immune-enhancing functional ingredient (by comparing the
expression levels of iNOS and COX2 in activated macrophages
treated with wheat germ extract simultaneously with LPS
compared to treatment with LPS alone, using Western blot
and qPCR).

Compared to red ginseng extract, wheat germ extract
demonstrated a significant increase in the production of iNOS
and COX2, which was directly proportional to the concentration.
This led to significant results related to immune indicators
associated with iNOS and COX2.

Group I: Normal control group (NC group)
Group II: Negative control - Cyclophosphamide treatment
(CY group)
Group III: Red ginseng treatment group (GS group)
Group IV: Wheat germ fermented extract group (WW group)
Group V: Wheat germ solvent extract group (WE group)

In animals, experiments are conducted by orally administering
specific substances or drugs to assess their effects on the
immune system or their ability to improve it.

To assess the effectiveness of NK cells in recognizing and
eliminating infected cells or cancer cells, thereby evaluating
the immune-enhancing effect of increasing NK cell activity.

Wheat germ extract exhibits NK cell activity-promoting effects
comparable to commercially available ginseng extract.
According to the results of ANOVA testing, its effectiveness
shows a linear or higher trend with increasing intake dose.

To evaluate the expression levels of specific cytokines by
measuring the activity level of the immune system,
which reflects the activity level of the immune system and
demonstrates its operational status.

Wheat germ extract is considered to contribute to improving
immunity, as it promotes an increase in immune-related
cells in vivo.