Comparative Analysis of Bioactive Components of Polygonatum cyrtonema Hua from Different Areas

WANG Dan ,ZHANG Hong ,LIU Jia-li ,MAN Qi ,DONG Xin-rong ,LIU De-ming ,HUANG Li-xin

1.College of Science,Hunan Agricultural University,Changsha 410128,PRC;2.Testing and Analysis Center,Hunan Agricultural University,Changsha 410128,PRC;3.Hunan Chongshuntang Technology Co.LTD,Changsha 410208,PRC

Abstract Polygonatum cyrtonema Hua from five different areas was used as experimental material,total saponins and total flavonoids.Colorimetric analysis method was used to compare those contents.The results showed that the contents of Polygonatum polysaccharides from different areas were in the range of 8.09%～11.78%,whereas the content of total saponins and total flavonoids was quite different,which were 2.73%～5.01% and 0.21%～0.71%,respectively.In general,these Polygonatum samples had higher polysaccharide content with obvious differences for the contents of total saponins and a low contents of flavones.

Key words Polygonatum;Polysaccharide;Total saponin;Total flavones

1.Introduction

As the dry rhizome of Liliaceae plant ofPolygonatumkingianumColl.et Hemsl,PolygonatumsibiricumRed.orPolygonatum cyrtonemaHua (Polygonatum cyrtonemaHua),Polygonatumcan nourish qi and yin,and invigorate spleen,lungs and kidney[1].Polygonatumwas first seen in TAO Hong-jing's "Supplementary Records of Famous Physicians" in the Jin Dynasty.It is stated in the "Collected Notes to Canon of Materia Medica" and "Compendium of Materia Medica":"Polygonatumalleviates depression to regulate qi,improves five internal organs,regulates the five internal organs,enriches muscles,strengthens bone marrow,doubles its power,and delays aging"[2],and now it is a dual-purpose plant for medicine and food[3].According to modern pharmacological and clinical studies,Polygonatumhas the functions of enhancing immunity,antibacterial,antiviral,anti-aging,anti-tumor,lowering blood sugar,lowering blood fat,and improving memory[4-9].

Polygonatum cyrtonemaHua is a genuine Chinese medicinal material in the southern region,but due to longterm mining,the wildPolygonatum cyrtonemaHua resources are scarce and cannot meet people's needs.Therefore,the artificially cultivatedPolygonatum cyrtonemaHua is emerging[10].However,the quality ofPolygonatum cyrtonemaHua is closely related to the variety and natural environment of the production area.At present,the Chinese Pharmacopoeia[1]qualitatively evaluates the quality ofPolygonatummainly,and only quantitatively evaluates the content ofPolygonatumpolysaccharides.Studies at home and abroad have shown thatPolygonatum cyrtonemaHua containsPolygonatumpolysaccharides,saponins and flavonoids and other biologically active ingredients[11-15].Therefore,a multi-index and simultaneous quantitative method should be adopted to objectively evaluate the quality ofPolygonatum cyrtonemaHua[16-19].The research team collected samples ofPolygonatum cyrtonemaHua planted in the wild in the forest of 5 areas in the South,and used the content ofPolygonatumpolysaccharides,total saponin,and total flavonoids ofPolygonatum cyrtonemaHua as indicators for quantitative comparison to provide a scientific basis for optimizing the local area condition to make it more suitable for the growth ofPolygonatum cyrtonemaHua.

2.Materials and Methods

2.1.Materials and reagents

Test materials:thePolygonatumused in the experiment was the tuber of artificially cultivated three-yearPolygonatum cyrtonemaHua.The collection time was in late November,and the collection locations were 5 main producing areas ofPolygonatum cyrtonemaHua Hunan,including Yongzhou area (A),Xiangxi area (B),Zhangjiajie area (C),Loudi area (D) and Sichuan Province (E),3 samples were collected from each area.

Reference samples:ginsenoside (B2p0234,Anhui Hefei Bomei Biotechnology Co.,Ltd.),sarsasapogenin reference substance (Batch No.:ST191029-010,Lemetian Medicine/Desite Biology),diosgenin reference substance (Batch No.:1539-200001,China Institute of Pharmaceutical and Biological Products),rutin (National Institute of Food and Drug Control).

Main reagents:glucose (AR),vanilline,anthrone,concentrated sulfuric acid,absolute ethanol,methanol,perchloric acid,glacial acetic acid,sodium nitrite,aluminum nitrate,sodium hydroxide,etc.,all of which are analytical reagents produced by Sinopharm Chemical Reagent Co.,Ltd.

Test instruments:UV-9600 UV-Vis spectrophotometer (Beijing Ruili Analytical Instrument Co.,Ltd.),AX-200 1/10 000 analytical balance(Shimadzu Philipines,Japan),DYF-200 Universal Crusher (Wenling Linda Machinery Co.,Ltd.),DZKW-D-2 constant temperature water bath (Beijing Ever Bright Medical Instrument Factory),RE-52C rotary evaporator (Shanghai Yarong Biochemical Instrument),SB25-12DT ultrasonic cleaner (Ningbo Scientz Biological Technology Co.,Ltd.).

2.2.Test method

2.2.1.Sample preparation

3 freshPolygonatum cyrtonemaHua samples from the same place were washed,then cut into about 1 mm slices and mixed thoroughly,and dried in an oven at 60℃ until the moisture content was less than 5%.After crushing with a universal pulverizer,the powder was collected in a sealed plastic bag,and stored in the refrigerator at 4℃ for later use.

2.2.2.Polygonatumpolysaccharides content analysis

2.2.2.1.Glucose standard curve

32.4mg of glucose monohydrate was weighed,put in a 100 mL volumetric flask,dissolved with water and diluted to the mark,and shaken well to obtain 0.294 5 mg/mL glucose standard preparation solution.0.1,0.2,0.3,0.4,0.5,0.6,0.8 mL of glucose standard stock solution were transferred into a 10 mL colorimetric tube with stopper,and made reach 2.0 mL by adding water,and shaken well.0.2% anthronesulfuric acid solution was added in ice water bath to make it reach scale slowly and mixed well.It was kept in a boiling water bath for 10 min,and cooled in ice water bath for 10 min after taking it out.With water as a blank control,the absorbance was measured at a wavelength of 582 nm.A standard curve was drawn with absorbance as the ordinate and glucose concentration as the abscissa.

2.2.2.2.Sample determination

The sample solution was prepared according to thePolygonatumpolysaccharides content determination method of Chinese Pharmacopoeia(2015 edition)[1].The color was developed and the absorbance was measured according to the above method,the concentration ofPolygonatumpolysaccharides was calculated according to the standard curve,and the content ofPolygonatumpolysaccharides inPolygonatum cyrtonemaHua was calculated according to formula (1).

Where,Ywas the content ofPolygonatumpolysaccharides or total saponin or total flavonoids,Cwas the concentration ofPolygonatumpolysaccharides or total saponin or total flavonoids (mg/L);Vwas the solution volume ofPolygonatumpolysaccharides or total saponin or total flavonoids (L);mwas the mass ofPolygonatum cyrtonemaHua (g).

2.2.3.Total saponin content analysis

2.2.3.1.Control sample selection

It refers to the method of literature[18].Specific operation:0.3 mL methanol solution,ginsenoside (1.07 mg/mL) solution,sarsaparilla saponin (0.65 mg/mL)solution and Diosgenin (0.27 mg/mL) solution ofPolygonatumextract were placed in four 10 mL cork tubes respectively,and the solvent was used up in a constant temperature water bath at 80℃.0.2 mL newly prepared 5% vanillin-glacial acetic acid solution was added,and then 0.8 mL perchloric acid was added under ice water bath.It was shaken well,and put into 60℃ constant temperature water bath pot for heating for 15 min.It was taken out and cooled in an ice-water bath for 2 min,and glacial acetic acid was added to make it reach 5 mL.It was shaken and left to stand for 5 min.An ultraviolet-visible spectrophotometer was used to measure the maximum absorption wavelength at 300～700 nm.

2.2.3.2.Draw the standard curve of diosgenin

2.7mg of diosgenin was weighed in a 10 mL volumetric flask,and methanol was added to dissolve and dilute to the mark.After shaking well,a stock solution containing 0.27 mg/mL of diosgenin was obtained.0.2,0.3,0.4,0.5,0.6 mL of diosgenin standard stock solution were transferred and placed in a 10 mL color-comparison tube for color development.The absorbance at 452 nm was measured with an ultraviolet-visible spectrophotometer.A standard curve was drawn with absorbance as the ordinate and concentration as the abscissa.

2.2.3.3.Determine the content of saponin in the sample

0.25g of the driedPolygonatumpowder was weighed and placed in a 250 mL round-bottomed flask.After adding 150 mL of 80% ethanol solution,it was treated by heat and reflux extraction for 1 h,and then filtered while it was still hot after cooling;the residue and filter paper were put back into the roundbottomed flask and extracted again and filtered.The filtrate was collected and concentrated to dryness under reduced pressure,and ultrasonically dissolved using the methanol.The volume was adjusted to 25 mL to obtain a sample solution.The 0.20 mL sample solution was removed into a color-comparison tube,and after the solvent was used up,the color was developed according to the above method and the light absorption value was measured.The total saponin concentration was calculated by standard curve,and the total saponin content inPolygonatum cyrtonemaHua was calculated by referring to formula (1).

2.2.4.Total flavonoids content analysis

2.2.4.1.Draw rutin standard curve

3.5mg of rutin was weighed in a 50 mL volumetric flask,and dissolved using 60% ethanol solution,and then diluted to the mark with 60%ethanol,and shaken well to obtain a rutin standard stock solution with a concentration of 0.07 mg/mL.It referred to the method of literature[19].0.5,1.0,2.0,3.0,4.0 mL of rutin standard stock solution were transferred into a 10 mL volumetric flask.Then 0.3 mL of 10% aluminum nitrate solution was added,and it was shaken well and kept stand for 6 min;then 2.5 mL of 4% sodium hydroxide solution was added,finally the volume was determined with 70% ethanol,and kept stand for 15 min.The absorbance at 510 nm was measured with the blank reagent as a control.A standard curve was drawn with rutin concentration as the abscissa and absorbance as the ordinate.

2.2.4.2.Determine total flavonoids content in samples 0.5 g of driedPolygonatum cyrtonemaHua powder was weighed into an erlenmeyer flask,and extracted ultrasonically for 20 min after 25 mL of 60% ethanol solution was added;the supernatant was poured into a centrifuge tube,and centrifuged at 3 000 rpm for 10 min,and the supernate was collected to obtain the sample solution.An appropriate amount of the sample solution was transferred into a 25 mL color-comparison tube,evaporated and concentrated to less than 5 mL in a constant temperature water bath at 95℃.After cooling,the color rendering was taken out,and the light absorption value was determined at 510 nm.The total concentration was calculated using the standard curve.The total flavonoids content inPolygonatum cyrtonemaHua was calculated further according to formula (1) .

3.Results and Discussion

3.1.Polygonatum polysaccharides

3.1.1.Glucose standard curv

The absorption value of glucose was determined at 582 nm after the color development of anthronesulfuric acid.The linear relationship between absorption value and glucose concentration was shown in Fig.1.

Fig.1 Standard glucose curve

Fig.1 showed that in the range of 2.945～20.615 mg/L,there was a good linear relationship between absorption value and concentration.The regression equation wasY=-0.019+0.042 42X,R=0.994,n=7.

3.1.2.Sample determination

It is generally believed thatPolygonatumpolysaccharides is the main biologically active component ofPolygonatum,and it is also the only quantifiable indicator component for evaluating the quality ofPolygonatumin the Chinese Pharmacopoeia[1].Therefore,comparing the content ofPolygonatumpolysaccharides is one of the basic elements to evaluate the quality ofPolygonatum.The determination results of the 5Polygonatum cyrtonemaHua from different areas were shown in Fig.2.

Fig.2 Polygonatum polysaccharides content of Polygonatum cyrtonema Hua from different Areas

It can be seen from Fig.2 that among the several samples investigated,thePolygonatumpolysaccharides content of D sample was the highest,whereas thePolygonatumpolysaccharides content of thePolygonatum cyrtonemaHua samples from the 5 origins can meet the requirements of the Chinese Pharmacopoeia for anhydrous glucose to be not less than 7.0% (calculated as dry product)[1].

3.2. Polygonatum saponin

3.2.1.Control sample selection

Modern research showed thatPolygonatum cyrtonemaHua contains bioactive substances of steroidal saponins and triterpene saponins[3,15].However,the Chinese Pharmacopoeia[1]currently only uses the TLC method for qualitative detection ofPolygonatumsaponins.In recent years,people began to pay attention to the quantitative evaluation of saponins inPolygonatum.Although HPLC method has the advantage of determining the content of single saponins[20-21],it is restricted by equipment and takes a long time to perform the task.In particular,different places of origin lead to great differences in the types and contents of saponins inPolygonatum cyrtonemaHua,and multiple control samples are needed to eliminate the interference of other substances when determining the total amount.Colorimetric analysis method for the determination of total saponin content of Chinese herbal medicines is fast and suitable for the comparison of total saponin of multiple samples,and its instrument is easy to be obtained.However,the Colorimetric analysis method does not have a unanimously recognized standard method for the determination of total saponin ofPolygonatum cyrtonemaHua,especially without a recognized control sample.At present,the control samples used in practice are ginsenoside[22]and dioscin[23].Some people think sarsasapogenin is better[24].In order to select the maximum absorption wavelength,the author referred to the methods mentioned above to conduct 300～700 nm wavelength scanning with the sample solutions of ginsenoside,dioscin and sarsasapogenin andPolygonatum cyrtonemaHua after color development.The results were shown in Table 1.

Table 1 The maximum absorption wavelength of saponin colorimetric analysis

It can be seen from Table 1 that the wavelength of the maximum absorption peak of the sample solution was close to that of diosgenin.Therefore,the author chose diosgenin as the control sample.

3.2.2.Content determination

Taking diosgenin as the control sample,the linear relationship between the absorbance and its concentration at 542 nm wavelength after color development was shown in Fig.3.

Fig.3 Standard curve of diosgenin

Fig.3 showed that the linear relationship between the two was good when the concentration of diosgenin was in the range of 5.4～16.2 mg/L.The regression equation was:Y=-0.005 2+0.088 04X,R=0.994,n=5.The total saponin content ofPolygonatum cyrtonemaHua sample was further determined,and the results were shown in Fig.4.

Fig.4 Total saponin content of Polygonatum cyrtonema Hua from different area

It can be seen from Fig.4 thatPolygonatumof sample A had the highest total saponin content,followed by E,and the other three samples were similar.

3.3. Polygonatum flavones

3.3.1.Standard curve line

Taking rutin as the control sample,after the color development by sodium nitrite and aluminum nitrate,the linear relationship between absorbance value and concentration at the wavelength of 510 nm was shown in Fig.5.

Fig.5 Standard curve line of rutin

It can be seen from Fig.5 that within the range of rutin concentration of 3.5～28.0 mg/L,the linear relationship between absorbance and concentration was good,and the regression equation was:Y=-0.002 05+0.001 09X,R=0.999,n=5.

3.3.2.Sample determination

SincePolygonatum cyrtonemaHua had a low content of flavones,after many preliminary experiments,the content of flavones inPolygonatum cyrtonemaHua was determined after the extracted samples were concentrated.The results were shown in Fig.6.

Fig.6 Total flavonoids content of Polygonatum cyrtonema Hua from different areas

The results in Fig.6 showed that,overall,the content of flavones inPolygonatum cyrtonemaHua was low,whereas there were also obvious differences among different producing areas.For the samples involved in the experimental study,the total content of samples E and C was high,while that of sample D was the lowest.

4.Discussions

Hunan is a genuine producing area ofPolygonatum cyrtonemaHua.In order to meet the planting needs of high-quality genuine Chinese medicinal material ofPolygonatum cyrtonemaHua,the research team collected three artificially cultivated samples ofPolygonatum cyrtonemaHua from the main producing areas ofPolygonatum cyrtonemaHua in Hunan,Zhangjiajie,Loudi,Yongzhou and Sichuan Province.And the contents ofPolygonatumpolysaccharides,total saponin,and total flavonoids were determined systematically,analyzed and compared.In terms of the samples collected in the experimental study,thePolygonatumpolysaccharides were the most abundant one in Loudi region.However,the content of the above samples all met the requirements of Chinese Pharmacopoeia.The content of total saponin was the highest in Yongzhou,while the content of flavones was the highest in Zhangjiajie and Sichuan.In general,the contents ofPolygonatumpolysaccharides and total saponin inPolygonatum cyrtonemaHua from the above 5 producing areas were relatively high,whereas the content of total flavonoids was generally low.

Polygonatum cyrtonemaHua is a perennial herb with the same medicine and food,and the market demand is increasing due to its good medicinal and edible value.In recent years,excessive mining of wildPolygonatum cyrtonemaHua has led to a rapid decline in production and damage to the ecological environment.As a shade loving plant,Polygonatum cyrtonemaHua is suitable for planting in the forest,especially for the needs of poverty alleviation and beautiful rural construction in poor mountainous areas.Therefore,the introduction and domestication ofPolygonatum cyrtonemaHua is emerging as a new type of underwood medicinal herb industry.The study analyzed and compared the contents ofPolygonatumpolysaccharides,saponin and flavones inPolygonatum cyrtonemaHua samples from 5 different producing areas,which can provide scientific basis for the introduction,domestication and standardized cultivation ofPolygonatum cyrtonemaHua in the South.In future studies,the research group will also further track the quality ofPolygonatum cyrtonemaHua in these regions,so as to provide more and more comprehensive scientific data for the development of a genuine Chinese medicinal material ofPolygonatum cyrtonemaHua in southern China.