Comparative Study on the Polysaccharide Contents and Antioxidant Activities of Hippophae rhamnoides subsp. sinensis and Hippophae gyantsensis

This study compares the polysaccharide content and the antioxidant activity between Hippophae rhamnoides sub. sinensis and the Hippophae gyantsensis to elucidate their differences. The study is methodologically sound, and it addresses a gap in the literature regarding species-specific differences in polysaccharide characteristics.

The findings of this study are significant for quality assessment and the potential pharmaceutical applications. To begin, transfer five grams of sea buckthorn sample powder into a 250 milliliter round-bottomed flask. Then add 50 milliliters of anhydrous ethanol, and soak it at room temperature of 25 degrees Celsius for 24 hours.

The next day, filter the soaked sample powder using a Buchner funnel to obtain the filter residue. Wash the filter residue three times with 50 milliliters of deionized water. Now, transfer the defatted filter residue into a dried 250 milliliter round-bottomed flask.

Add 150 milliliters of deionized water at a solid to liquid ratio of one to 30 grams per milliliter. Perform reflux extraction in a water bath at 80 degrees Celsius for two hours twice. Then concentrate the extract using a rotary evaporator.

Store the concentrated solution in a refrigerator at four degrees Celsius for later use. Next, add Sevag reagent to the crude polysaccharide solution in a one to four volume ratio and place on a magnetic stirrer. Then transfer the mixture into a 50 milliliter centrifuge tube.

Centrifuge the suspension at 4, 000 g for 15 minutes, and collect the supernatant. To concentrate the polysaccharide extract after protein removal, add anhydrous ethanol at a volume ratio of one to three to precipitate polysaccharides. Place the mixture at four degrees Celsius overnight.

On the next day, remove the upper layer of ethanol. Transfer the mixture into a 50 milliliter centrifuge tube and centrifuge as before. Freeze dry the precipitate to obtain total polysaccharides.

Transfer three grams of phenol into a brown volumetric flask. Add 50 milliliters of deionized water to dissolve the phenol. Shake until homogeneous, then label and store flask for later use.

Next, transfer 10 milligrams of anhydrous glucose into a 10 milliliter volumetric flask. Add deionized water and shake the mixture thoroughly. Then label the flask.

For the preparation of the test sample solution, weigh five milligrams of freeze dried sea buckthorn polysaccharides into volumetric flasks. Add deionized water to bring each to a final volume of 50 milliliters. Sonicate each mixture for 20 minutes until completely dissolved.

Dilute the standard glucose solution with deionized water to 0.1 milligrams per milliliter. Then using a micropipette transfer 200, 300, 400, 500, 600, and 700 microliters into six clean test tubes. Add pure water to bring each to a total volume of one milliliter, and gently mix each solution to ensure uniform dilution.

Add one milliliter of 6%phenol solution and five milliliters of sulfuric acid to each test tube and mix well. Transfer the samples into cuvettes. Use pure water as blank and measure absorbance at 490 nanometers with a UV visible spectrophotometer in three replicates.

For the precision test, prepare the test solution. Perform phenol-sulfuric acid colorimetry. Determine absorbance and repeat six times to calculate relative standard deviation.

For the stability test, prepare the test solution. Perform colorimetry at zero, two, four, six, eight, and 10 hours post-preparation. Determine absorbance at the aforementioned time points to calculate relative standard deviation.

For the repeatability test, prepare six parallel replicates of the sample. Conduct phenol-sulfuric acid colorimetry, and determine absorbance following the established procedure. To perform the recovery test, prepare nine parallel replicates of the test solution.

Add reference volumes equivalent to 80, 100, and 120%of total polysaccharide content. Then perform phenol-sulfuric acid colorimetry. For content determination, prepare the sample solution.

Perform phenol-sulfuric acid colorimetry following the established method. Then determine absorbance. Weigh 2.7 milligrams of DPPH into an empty 50 milliliter volumetric flask.

Add 50 milliliters of anhydrous ethanol to dissolve the DPPH and prepare a stock solution. Next, dissolve five milligrams of vitamin C in 10 milliliters of deionized water to prepare a 0.5 milligram per milliliter solution. Dilute this solution successively to prepare vitamin C test solutions.

Now dissolve five milligrams of each sea buckthorn polysaccharide sample in 10 milliliters of deionized water to prepare a 0.5 milligram per milliliter solution. Dilute these solutions successively to obtain test concentrations. Using the DPPH solution as the substrate, prepare the test groups in a 96 well plate.

Add 150 microliters of water and 50 microliters of DPPH for the negative control group, A0.Add 150 microliters of the sample or vitamin C solution and 50 microliters of DPPH for the sample group A1.For sample control group A2, add 150 microliters of sample and 50 microliters of anhydrous ethanol. Allow the reaction to proceed in the dark for 30 minutes. Then measure the absorbance at 517 nanometers.

The standard curve of glucose showed a strong linear relationship between absorbance and concentration in the range of 0.02 to 0.07 milligrams per milliliter. The average polysaccharide yield of Hippophae gyantsensis was significantly higher than that of Hippophae rhamnoides subspecies sinensis. The precision test demonstrated minimal variation in absorbance across six measurements, and RSD of 0.21%indicating high instrument precision.

The stability test showed that absorbance values remained consistent over a 10-hour period, with RSD value of 0.45%confirming the sample solution stability. The repeatability test produced consistent absorbance values across six replicates with RSD of 0.5%validating the method's repeatability. The recovery experiment for glucose content yielded an average recovery rate of 99.18%confirming the accuracy of the method.

The average polysaccharide content of Hippophae rhamnoides subspecies sinensis was significantly higher than that of Hippophae gyantsensis. Hippophae gyantsensis exhibited slightly higher antioxidant activity than Hippophae rhamnoides subspecies sinensis at all tested concentrations. At 0.5 milligrams per milliliter, the DPPH clearance rate of Hippophae gyantsensis reached 81.93%while Hippophae rhamnoides subspecies sinensis reached 75.17%