THE INVESTIGATION FOR BEE-QUEEN-LARVA PROTEINS

OUYANG Li1, LIU Ling2 and ZHAO Jing3

1The School of Public Health, Peking University, 100083; 2Medical and Health Analytical Center, Peking University, Beijing 100083 P. R. ) 3Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Xiangshan, Beijing 100093 P. R. China

Abstract: This paper made some investigations for the proteins of bee-queen-larva .The proteins from different extract liquid in several buffers with different pH were investigated by TOFMS and HPLC. 16 kinds of proteins with different molecular weight were detected within the mass range of 2kDa-20kDa.The proteins extracted with different extracting liquids were different in quantities and sorts. In addition, SOD were detected in bee-queen-larva. Fresh bee-queen-larva produced on the same year contains SOD activity of 310U/g, higher than that of honeycomb（85U/g）. This proved that bee-queen-larva contains activity material with anti-aging function. keywords: bee-queen-larva; protein; SOD; TOFMS; HPLC

Bee-larva is mainly generally recognized as larva of bee-queen. its volume is big and the nutrients ,medical efficacy are good which has very high value for the development.Bee-larva is the by-product in the production of royal jelly, generally 0.2-0.3 Kg Bee-larva can be gotten after production of 1 Kg royal jelly. The nutrient value of Bee-larva is very high, its main ingredients is similar with royal jelly and the content of protein is as high as 15.4%[1] which can become a natural resource of insect protein. Its efficacy for health maintenance was known in China since ancient time. An ancient Chinese medical book records that bee-larva has little sweet smell, same cold, no poisoning, treating headache and parasitic disease. Long eating people can become buxom, not old. Modern medicine has improved that bee-larva has the efficacy of invigoration and rejuvenescence and aid effect for disease treatment. Although Bee-larva’ s nutrient and health maintenance effect is well known but the investigation for its chemistry and ingredients is very little[2,3]。For the better use of bee-larva resource, the paper made some investigations for the proteins of bee-larva and its anti-aging function ingredient—SOD.

Experimental Section 1. Instrument and Equipment Mass spectrograph and the condition: LD1700 TOF-MS； Ionizing mod: MALDI； Matrix：Sinapinic acid；Mass range：2KDa-20KDa。 Chromatograph and condition： P-4000 HPLC from TSP Co. Ltd, column：Protein PAK125 300×7.8 mm ；UV detector；wave-length：280 nm； solvent：0.9%NaCl/water solution；flow rate：1.0 mL/min。 TU-1800spcUV spectrophotometer；detect wave-length：525 nm。

Proceedings of the 37th International Apicultural Congress, 28 October – 1 November 2001, Durban, APIMONDIA 2001 To be referenced as: Proc. 37th Int. Apic. Congr., 28 Oct – 1 Nov 2001, Durban, South Africa ISBN: 0-620-27768-8 Produced by: Document Transformation Technologies Organised by: Conference Planners 2 Resource of sample bee-larva：fresh bee-larva produced in this year from our institute’s bee mill，preserved at –18 。 Royal jelly: produced in the same year from our institute’s bee mill，preserved at -18℃。

3 reagents and medicine： standard sample of SOD was purchased from Sigma；all the reagent are analytical purity.

4 SOD standard solution： 50U/mL SOD standard solution in phosphoric salt buff。preserved at 4 ℃，used within 30 days。

5 extracting solvents (1) Barbital sodium-hydrochloride buff solution，pH 8.2（B） (2) Tris-hydrochloride buff solution，pH 8.2（T） (3) Citric acid –sodium citrate buff solution，pH 4.9（C） (4) Ammonia sulfate saturated solution，pH 7.2（S） (5) Acetone (A） (6) 95% ethanol（E）

6 preparation for bee-larva extracting solution and measurement weighing 1g fresh bee-larva and put it on glass homogenizer，adding extracting solution 1mL， homogenating for 1 minute. Transform it to a centrifuge tube，centrifuging for 15 minutes （4000 rpm）。The superior or middle solutions are protein extracting solution。Making HPLC and MS measurements for each extracting solution under the working condition mentioned above。 Making the SOD [4] activity measurement for bee-larva extracting solution extracted with extracting solvents B,C,T and royal jelly water solution(50%) separately according to processes in table 1 (hydroxylamine method).

TABLE 1: SOD MEASUREMENT PROCESS solvents Sample tube Control tube （mL） （mL） Phosphatic buff（pH 7.8） 1.0 1.0 Bee-larva extracting solution（or SOD standard 0.04（0.02） - solution） 10 m mol/L hydroxylamine hydrochloride 0.2 0.2 7.5 m mol/L xanthine 0.2 0.2 0.023U/mL xanthine oxydase 0.2 0.2 Distilled water 0.5 0.5 Mixing in 37℃ warm water bath for 30 minutes 3.3g/L sulfanilic acid 2.0 2.0 1.0g/L α- naphthylamide 2.0 2.0 mixing，standing at room temperature for 15 minutes， adjusting zero with phosphatic buff and measuring SOD at 525 nm。

Results and discussion 1 the comparison of bee-larva protein extracted by different extracting solvents measuring the different pH buff extracting solutions and extracting solutions extracted by 95% ethanol and acetone , the results is on the table 2. by the retention time of HPLC peak, 14 kind of different protein can be observed from 6 kind of extracting solution. As some peaks are overlapping the number of the protein should be more.

Table 2 results of HPLC measurement Peak No. 1 2 3 4 5 6 7 8 extracting RT 8.66 9.24 12.07 13.59 solutionB area% 2.19 38.96 51.60 6.95 extracting RT 8.46 11.05 12.28 12.69 14.14 14.88 16.80 solution T area% 1.20 4.23 39.47 44.20 1.25 8.26 1.15 extracting RT 8.43 10.96 11.81 12.42 12.98 14.00 14.83 solutionC area% 9.38 15.90 33.54 27.02 5.78 1.02 5.88 extracting RT 6.63 8.52 11.03 12.31 13.09 14.96 solutionS area% 7.07 18.98 2.57 56.44 6.79 7.57 extracting RT 8.41 11.01 12.01 12.39 12.96 14.07 14.75 19.01 solutionA area% 13.19 10.24 33.75 13.92 8.68 1.73 2.90 13.33 extracting RT 8.41 11.01 12.27 12.96 14.26 14.78 19.13 19.74 solutionE area% 5.90 6.78 65.89 7.02 1.65 7.32 1.58 3.66

Recently the technique of bio mass is developing very fast, and wildly used in the fields of analysis, biochemistry and life sciences. Its character is high-selectivity, high-sensitivity and high flux. MALDI-TOF-MS has very protruded superiority in analyzing protein, especially mixed protein. We injected mixtures directly and with MALDI-TOF-MS to analyze every bee-larva extracting solutions. the molecular weight of every protein were direct obtained，the results in on the table 3。Within the range of 2kDa—20kDa，16 kind of proteins were observed from 6 kinds of extracting solutions.。

Table 3 : results of MS measurement extracting Molecular weight Number of solution proteins B 3144 4807 5572 6484 6684 13870 15489 7 T 3146 4188 5065 6828 9155 10321 13937 15666 8 C 3135 4103 5562 8361 13762 15500 6 S 3161 4831 4997 5614 7060 7810 8290 14000 8 A 3115 4753 5657 8354 10350 13812 6 E 3139 5584 6627 10439 13756 15533 6

From the results of HPLC and MS, we observed that in different extracting solutions the number, kinds and quantitative of the proteins are different. When one single protein is demanded, one special solvent could be selected in odor to obtain the better results.

2 the measurement for SOD of the bee-larva extracting solutions SOD belongs to metalloenzyme，comprising of metal elements and protein。SOD catalyses the - - differentiation of superoxide anion （O2 ）to H2O2 and O2，precluding the toxicity of O2 to cell, with efficacy of anti-aging[5].. The SOD measurement results for bee-larva extracting solutions and royal jelly solution as blank are in the table 4. the SOD contents of 3 kinds of bee-larva extracting solutions measured are all higher than royal jelly，indicating in bee-larva’s SOD is very rich. This results is coincidence with record of Chinese ancient book ----“bee-larva has efficacy of cosmetology and anti-aging.

table4 SOD measurement results extracting extracting extracting Royal jelly solution B solution C solution E SOD(%) inhibiting rate 47.3 53.7 50.7 13.8 SD 6.4 4.8 0.9 1.3 RSD 13.6% 8.9% 1.8% 9.7% SOD activity （U/g） 290 330 311 85

Reference: 1 Cheng like et al.: modern Apiculture and processing of bee’s production 2 Zhang jian et al.: Journal of the bee，1995，10：6。 3 Zhao jing et al.: Journal of the bee，1995，1：7。 4 Supervision law collection for health food （1997）。 5 Fang Yunzhong et all., basic theory of free radical and enzyme and their application in biology and medical science（1989）71.

THE INVESTIGATION FOR BEE-QUEEN-LARVA PROTEINS

OUYANG Li1, LIU Ling2 and ZHAO Jing3

1The School of Public Health, Peiking University, Beijing 100083; 2Medical and Health Analytical Center, Peiking University, Beijing 100083 P. R. China) 3Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Xiangshan, Beijing 100093 P. R. China

RESUME

Ouyang Li: female, The School of Public Health, Peiking University Beijing , 100083 P. R. China. Obtained bachelor of engineering sciences degree in Jan. 1982. Beijing Chemical Engineering Institute, PR China. Obtained master of sciences degree in March 1990, Osaka university, .

Liu Lin: female, Medical and Health Analytical Center, Peiking University, Beijing 100083 P. R. China. E-mail: [email protected]. Obtained BS degree in 1983, Department of Chemistry, Sichuan University.

ZHAO Jing: female , Associate Professor, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Xiangshan, Beijing 100093 P. R. China Executive Director, Bee Production Quality Supervisory Inspection and Test Center (Beijing) of Agriculture Ministry China.