The kernel of Carya cathayensis Sarg was purchased in Lin’an, Hangzhou Province China and store at 4˚C for use. All chemicals and standards were purchased in aladin company (China), and all standards were bought in Sigmaaldrich company (USA).

The content of the total fatty acids was evaluated by Pritam method with slight modification [18] . The 3 g of samples in soxhlet extractor were extracted with petroleum ether at 90˚C for 8 h. The extracted solutions were condensed to 1 - 2 mL with rotary evaporator under vacuum and dried at water bath till no solvents left. The residues were continually dried at 105˚C for 1 h and weighted. And repeat dried step for several times till to constant weight (within 2 mg difference in followed two dried). The contents of fats were calculated as follows:

X = m 1 − m 0 m 2 × 100

X―Contents of fat in samples (g/100g)

m₁―the weight of flask and fat (g)

m₀―the weight of flask (g)

m₂―the weight of samples (g)

100―conversion coefficient

The Micro-Kjeldahl method was adopted for determination of total proteins with little modification [19] . With addition of 0.4 g copper sulfate, 6 g potassium sulfate and 20 mL sulfuric acid, 2 g of solid samples were digested at 420˚C for 1 h, then cooled to room temperature. And 50 mL water were added for the evaluated. The total proteins were determined as follows:

X = ( v 1 − v 2 ) × c × 0.0140 m × v 3 × 100 × F × 100

X―Contents of proteins in samples

v₁―Sulfuric acid volume consumed in samples (mL)

v₂―Sulfuric acid volume in blank samples (mL)

C―Contents of sulfuric acid (mol/L)

0.0140―Mass of nitrogen that 1 mL sulfuric acid (c = 1 mol/L) comsumed (g)

m―Mass of samples (g)

v₃―Volume of digested (mL)

F―Conversion of Nitrogen to protein

100―Conversion coefficient

Samples were firstly heated in oven till no smoking, then calcinated in muffle furnace at 550˚C for 4 h and cooled to 200˚C. The samples were weighted after put into drier for 30 min repeatedly, calcinated till the weight of the sample to constant weight (within 0.5 mg difference).

X = m 1 − m 2 m 3 − m 2 × 100

X―Ash content of samples

m₁―Weight of crucibles and ash (g)

m₂―Weight of crucibles (g)

m₃―Weight of crucibles and samples (g)

The samples placed in weighting flasks were dried at 105˚C for 3 h, then cooled to room temperature in drier. Followed, weighted and repeatedly dried every 1 h till the adjacent 2 times being within 0.0005 g of difference.

X = w 1 − w 2 w 1 − w 0 × 100

X―Contents of moisture

w₁―Weight of wet samples and weighting flasks

w₂―Weight of dried samples and weighting flasks

w₀―Weight of dried weighting flasks

Methyl esterification: With addition of 2 mL benzene-petroleum ether (1:1 v/v) 0.1 g of oil samples solved in 10 mL tubes stood for 5 min, then added 1 mL 0.1 mol/L KOH of methanol solution. The mixture was stood for 10 minutes and then added saturated NaCl solution to 10 mL of constant volume.

Gas chromatographs spectrometry (GC) was conducted on a Hewlett-Packard (HP) 7890 GC coupled to a FID Detector using a 30 m × 0.32 mm id., 0.25 μm film thickness DB-WAXETR capillary column (J & W Scientific Inc., Folsom, CA) with nitrogen as carrier gas. (150˚C for 2 min and then programmed to 190˚C at 5˚C/min and held for 20 min).

Starch standard was boiled in distilled water, cooled and made up to a measured volume with distilled water. So that a 2.0 mL aliquot contains 10 to 200 micrograms of starch. Aliquot in a colorimeter tube was treated with 4.0 mL of 0.1% anthrone in concentrated sulfuric acid (4 h to 9 days) rapidly added from a pipet or burette. The solution was mixed immediately and allowed to air cool. After approximately 10 to 15 min, the tube is cooled completely in a cold-water bath. At the same time one or more 2.0 mL starch standards (100 micrograms suitable) received the same treatment. The colorimeter was adjusted to zero with the reference blank and the samples and standards are then read. When a logarithmic scale colorimeter was used, concentrations were proportional to scale readings. The pro ortionality constant is determined by the standards used.

1.0 mg/mL of the 23 kinds of standards solutions (0.5 mL) were prepared by dissolving the standards in methanol and diluted into different contents in methanol and acetic acid.

The diol-bonded phase cartridge was conditioned according to the method described by Garcia et al. (García, Yousfi, Mateos, Olmo, & Cert, 2001). 2.5 g of the oil samples were dissolved in 6 mL hexane. Then, the samples were loaded to the cartridges, and washed by 6 mL hexane, 3 mL of hexane/ethyl acetate (90:10, v/v), followed extracted with 10 mL of methanol and evaporated at 40˚C under vacuum, dried away of the solvent to obtain the solid residues. The residues were dissolved in 2 mL methanol and water (1:1, v:v), finally filtrated through 0.45 μm membrane.

A filtered aliquot (20 μL) of the final colorless solution was injected onto the HPLC system (an Agilent Technologies 1100 liquid chromatographic system equipped with an SPD-10A UV detector). The column was a WondaSil C18 reverse column (4.6 mm × 250 mm, 5 μm particle size) maintained at 30˚C. The gradient elution, at a flow rate of 1.0 mL/min, was achieved using the following mobile phases: water as solvent A, 0.2% acetic acid in water as solvent B. The solvent gradient was programmed as follows: from 10% (A) in 5 min, to 20% A in10 min, to 30 A% in 20 min, to 40% A in 35 min, to 60% A in 45 min, to 70% A in 55 min, to 100% A in 70 min, followed by 5 min of maintenance.

Dissovled in 2 mL of benzene-petroleum ether (1:1, v:v), 0.1g kernel oil of Carya cathayensis Sarg was added into 2 mL of 0.4 M KOH in methanol solution. Followed the mixture was washed by saturated NaCl solution, and the upper layer was collected and centrifuged at 2000 r/min for 10 min.

Gas chromatographs spectrometry (GC) was conducted on a Hewlett-Packard (HP) 7890 GC coupled to a FID Detector using a 30 m × 0.32 mm id., 0.25 μm film thickness SP-2560 capillary column (J & W Scientific Inc., Folsom, CA) with nitrogen as carrier gas at a rate of 1.0 mL/min (160˚C for 5 min and then programmed to 220˚C at 5˚C/min and held for 15 min).

1.0 g oil samples were dissolved in 10 mL hexane, and 1.5 mL of the mixture were transferred to 2 mL centrifuge tub. And centrifugated for 10 min at 10,000 r/min and filtrated by 0.45 μm organic membrane.

The HPLC system (Agilent1100, USA) used consisted of a G1311A liquid chromatograph pump, G1379A online degas unit, G1313A automatic injector with a 10 μL loop, and fluroescence detector. The data were collected using an Agilent1100 ChemStation system provided by Agilent Company. A special ZORBAX Eclipse XDB-C18 HPLC column (250 mm × 4.6 mm，5 µm), (Agilent, USA), optimized for the separation of vitamin E, was used at ambient temperature of 30˚C. The vitamin E elution was performed with a mixture of n-hexane and isopropanol in a ratio of 97:3 (v/v) at a flow rate of 1 mL/min, and UV absorbance of the effluent was monitored at 333 nm and scan time was 7 min. The vitamin E was quantified by comparing their integration values of peak area to a calibrated standard curve.

1.0 g oil sample dissolved in 2 mol/L KOH ethanol solutions was saponificated at 85˚C water bath for 1 h. After saponification, the mixture was transferred into 500 mL funnel and added 50 mL saturated NaCl solution, and extracted 3 times with 50 mL petroleum ether, then washed the organic phase with deionized water to neutral. Followed, the organic phase was dried by anhydrous NaSO₄, removed solvent, dissolved in 10 mL n-hexane and filtrated for application.

Gas chromatographs spectrometry (GC) was conducted on a Hewlett-Packard (HP) 7890 GC coupled to a FID Detector using a 30 m × 0.32 mm id., 0.25 μm film thickness HP-5 capillary column (J & W Scientific Inc., Folsom, CA) with nitrogen as carrier gas at a rate of 1.0 mL/min. (160˚C held for 1 min and then programmed to 280˚C, held for 5 min then at rate of 5˚C/min to 300˚C and held for 15 min). The sterol was quantified by comparing their integration values of peak area to a calibrated standard curve.