*3.4.1.2 Analysis of 10-HDA in RJ*

10-Hydroxy 2-decenoic acid (10-HAD) belongs to the important physiologically active components of RJ. Analysis of 10-HDA in RJ was performed by the ordinary method (Japan Health And Nutrition Food Association, 2012).

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping… DOI: http://dx.doi.org/10.5772/intechopen.89647*

### *3.4.1.2.1 Preparation of sample solution*

4.Watering trays for honeybees are set around the apiary and the beehive.

6. Second-instar larvae are used for larva transfer to artificial queen cell cups for

5.Harvested honey is always stored in a cool, dark place.

8.The number of artificial queen cell cups is to be 100 at most.

10.Both honey and RJ are always filtered at the apiary.

13. In principle, honey is prepared from a single nectar plant.

and later crops of high purity are released as product.

15.Concentration by heating is not performed.

For each sample, 60 g was harvested as follows:

harvest after 48 h was used for analysis.

9.The work rotation rate of honeybees is to be set at a constant 25%.

11.The old beehive and hive frame are disposed of, and new ones are used.

14.For harvesting honey, "morning squeeze" is performed instead of "evening squeeze", and honey matured in the hive cells is harvested. Only the fourth

In Qing hai Haibei Menyuan, RJ was harvested from rape blossoms (almost in full bloom) using Occidental honeybees (*Apis mellifera*) for 15 days from

1.Natural Beekeeping (rotation harvest giving a rest to honeybees): Using 96 artificial queen cell cups in each colony, second-instar larvae were grafted into artificial queen cell cups. RJ was harvested 48 h after larva transfer. After giving a two-day (48-h) rest to the honeybees, larva transfer was performed, and harvest was performed again after 48 h. Similarly, a two-day (48-h) rest and harvest after 48 h were performed again, and the RJ obtained in the third

2.Ordinal beekeeping (continuous harvest giving no rest to honeybees): Using 96 artificial queen cell cups in each colony, second-instar larvae were grafted into artificial queen cell cups. RJ was harvested 72 h after larva transfer. Immediately after harvest, larva transfer was performed and harvest was

performed again, and the RJ obtained in the third harvest after 72 h was used

10-Hydroxy 2-decenoic acid (10-HAD) belongs to the important physiologically active components of RJ. Analysis of 10-HDA in RJ was performed by the ordinary

performed again after 72 h. Similarly, the same procedures were

method (Japan Health And Nutrition Food Association, 2012).

7.RJ is stored in a refrigerator set at 2°C.

*Modern Beekeeping - Bases for Sustainable Production*

12.Absolutely no antibiotics are used.

RJ production.

July 10–24, 2010.

for analysis.

**136**

*3.4.1.2 Analysis of 10-HDA in RJ*

After homogenizing the sample RJ, 0.5 g of RJ was weighed accurately into a 200 ml beaker. Water 80 ml was added and the mixture was stirred vigorously until a uniform suspension was obtained. Next, methanol 80 ml was added, and the mixture was further stirred for 20 min and transferred into a 200 ml measuring flask. The beaker was washed with a mixture of water and methanol (1:1), and the washing was added to the 200-ml measuring flask. The mixture of water and methanol (1:1) was added to the measuring flask to make exactly 200 ml. The resulting fluid was filtered with a membrane filter 0.45 μm in pore size, and the filtrate was used as the sample solution.

### *3.4.1.2.2 Preparation of standard solution*

Exactly 0.01 g of 10-HDA Reference Standard was weighed accurately and dissolved in methanol to make 50 ml. Exactly 5 ml of this solution was added with the mixture of water and methanol (1:1) to make 20 ml. The resulting solution was used as the standard solution.

#### *3.4.1.2.3 Quantitative analysis*

The test was performed with 10 μl each of the sample solution and the standard solution as directed under the Liquid Chromatography in line with the following conditions. The areas of 10-HDA peaks from both solutions, At and As, were determined, and the amount of 10-HDA in the sample was calculated according to the following formula.

Amount mg ð Þ of 10 � HDA in 100 g of sample ¼ weighed amount mg ð Þ of 10 � HDA Reference Standard � At*=*As � 100 g*=*weighed amount gð Þ of sample

where As is the area of 10-HDA peak from the standard solution and At is the area of 10-HDA peak from the sample solution.

#### *3.4.1.2.4 HPLC operating conditions*

Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Column: An ODS column (4.6 mm<sup>Φ</sup> � 150 mm).

Column temperature: A constant temperature of about 40°C.

Mobile phase: A mixture of diluted phosphoric acid (1 in 1000) and methanol (1:1).

Flow rate: Adjusted so that the retention time of 10-HDA was in the range of 14–16 min.

Reproducibility: When the test was repeated six times with the standard solution in line with the above conditions described above, the relative standard deviation of 10-HDA peak areas was not more than 2.0%.

#### *3.4.1.3 Analysis of MRJP-1 Multimer in RJ*

MRJP-1 is a major protein of RJ. It is a multifunctional pharmaceutically important protein [24–26] and serves as a marker of the authenticity and quality of honeybee products [27, 28]. The analysis of MRJP-1 multimer in RJ was performed by the method reported previously (partially modified) [29].

The RJ was suspended in water. The supernatant obtained after centrifugation of the suspension was subjected to HPLC analysis.
