huangqi can tonify qi , and yinyanghuo can support yang ; the two are commonly used in chinese clinical medicine in the treatment of qi deficiency , yang insufficiency , and other diseases .
they can also promote osteoblast function , increase calcified bones , and promote dna synthesis of bone marrow cells ; an in vitro experiment has shown that yinyanghuo can promote osteoblast proliferation and differentiation .
huangqi yinyanghuo group ( hyg ) is the traditional chinese medicine which is used in clinical practice widely [ 2 , 3 ] .
over 90% of tumor cells have telomerase activity , while the majority of somatic cells lack or even do not have telomerase activity .
telomerase synthesizes telomeric dna repetitive sequences by reverse transcription with its own rna as the template and adds them to the telomere ends , in order to compensate for the terminal bases lost due to cell division and ensure the stability and integrity of chromosomes , thus providing the basis for eternal life of tumor cells .
htert is its rate - limiting component , expression of htert genes plays a decisive role in telomerase expression , and bcl-2 gene ( i.e. , b - cell lymphoma / leukemia-2 gene ) is a type of protooncogene which is capable of suppressing apoptosis .
htert and bcl-2 protein are both very good targets for cancer therapy . by studying the effects of hyg on htert and bcl-2 protein in osteosarcoma cells ,
this experiment provides a theoretical basis for clinical application of hyg in treatment of osteosarcoma .
agilent 1100 series hplc system ; kq-300 ultrasonic cleaner , nanjing kunshan ultrasonic instruments co. , ltd .
huangqi and yinyanghuo crude drugs ( proportion of the two herbs was 1  :  1 in this compounded prescription ) were purchased from guangzhou kelaimente medicine company , which were identified by the institute of botany of the guangzhou academy of sciences as the dried root of astragalus membranaceus ( fisch ) bge .
mongholicus ( bge . ) hsiao of the family leguminosae and epimedium brevicornum maxim . of the family berberidaceae .
icariin reference substance ( batch number 130526 - 152369 ) was obtained from guangzhou institute of biological products assay ; the water used was double distilled water , all the detection reagents were of hplc grade , and other reagents were all of chemical grade .
human osteosarcoma cell lines ( hos and cck8 ) were purchased from nanjing dingsheng biotechnology co. , ltd . fetal bovine serum ( hyclone ) was purchased from beijing saiaoruite biochemical products co. , ltd .
htert antibody was purchased from santa cruz biotechnology ; bcl-2 antibody , dab chromogenic reagent kit , and immunohistochemical staining kit ( sp kit ) were all purchased from chengdu hengyuan bioengineering co. , ltd .
investigation was performed on three levels of four factors : amount of water addition ( a ) , soaking time ( b ) , extraction time ( c ) , and extraction times ( d ) by orthogonal design table l9(3 ) using icariin as the indicator .
the chromatographic column used was hypersil ods2 ( 4.6  mm  250  mm , 5  m ) , mobile phase was methanol-0.38% phosphoric acid solution ( 48  :  52 ) , flow rate was 1.0  ml / min , detection wavelength was 270  nm , column temperature was 40c , and sample injection volume was 10  l .
icariin reference substance was accurately weighed and added to 70% methanol solution to prepare 0.01  mg / ml icariin reference solution ; after filtration , 2.00  ml of filtrate was precisely drawn , placed in a 50  ml flask , added to 40  ml of 70% methanol solution , ultrasonicated for 10  min , allowed to cool and stand at room temperature , then diluted to the mark by addition of 70% methanol solution , shaken uniformly , and filtered with 0.45  m membrane , and the subsequent filtrate was collected as the test solution .
reference solution was taken and prepared into solutions with concentrations of 0.010 , 0.020 , 0.050 , 0.100 , 0.200 , and 0.500  g / ml , respectively ; 10  l of the solutions was taken , and their peak areas were measured ; standard curves were plotted with the peak area as ordinate ( y ) and the icariin concentration as abscissa ( x ) , and regression equation was calculated to be y = 2428x  0.6658 , r = 0.9999 .
the results showed that the icariin concentration had a good linear relationship with the peak area within the concentration range of 0.01~0.50  g / ml ; see figure 1
. 10  l of reference solution was precisely drawn , sample was injected 5 times in parallel , peak area value of icariin was measured , and rsd value was calculated to be 1.52% , which indicated good precision of the instrument . test solution was prepared according to the test solution preparation method , sample was injected after standing for 0 , 2 , 4 , 8 , and 12  h , respectively , peak area of icariin was measured , and rsd value resulted to be 2.50% , which indicated that the test solution had good stability within 12  h. test solutions were prepared in quintuplicate according to the test solution preparation method , and peak areas were measured ; rsd of content was calculated to be 1.83% , which indicated good reproducibility of the present method .
approximately 0.20  g of samples with known concentration was accurately weighed in quintuplicate , placed in stoppered erlenmeyer flasks , and added precisely with 2  ml of 0.198  mg / ml icariin solution in dilute ethanol , and peak area was measured according to the method under sample assay , content was determined , and average recovery rate resulted to be 100.5% , with rsd value of 1.72% .
crude drugs were extracted according to the orthogonal design table l9(3 ) , extracts were combined , appropriate volume of extract was taken , and icariin content in the extract was determined according to the method under  plotting of standard curves  ; the results and analysis are shown in tables 2 and 3 .
it can be judged according to the results in table 2 that the size of influence of various factors on icariin extraction was d > a > b > c in descending order ; optimal extraction process was a3b1c2d3 , that is , addition of a 12-fold volume of water to the quangqi and yinyanghuo crude drugs with a weight ratio of 1  :  1 , soaking for 15  min , and three times of extraction with each time lasting 1.0  h ; the residues were then discarded and the decoctions were combined to give the extract . as can be seen from table 3 , factor d has a significant influence on extraction of icariin , while factors a , b , and c have relatively small influences within the investigation range , which presented no statistical significance .
the above screened optimal extraction process was validated , and the validation results are shown in table 4 . according to the results in table 4 , average content of icariin in the extract is 69.07  g , which is higher than the content in any test number , and does not have relatively large deviation as well .
cells were subcultured in an incubator set at 37c , 5% co2 , and saturated humidity ; subculture medium was 10% fbs and 100  /ml double antibody - containing high glucose dmem ( containing phenol red ) .
cells were seeded in 96-well plates at 5  10 cells per well . after culturing for 24  h ,
each well was filled separately with 200  l of hyg - containing media with final concentrations of 0.125 , 0.25 , 0.5 , 1 , 2 , 4 , and 8 
mol / l ; meanwhile , a nondrug treatment group was set up ; after incubation it was continued in the incubator for additional 24  h , 48  h , 72  h , 96  h , and 120  h , and each well was filled with 20  l of cck-8 solution and incubated in the incubator for another 4  h. absorbance of each well was measured at 490  nm wavelength using elisa reader ; cell morphology was observed under a microscope and photographed .
cell growth curve was plotted with time as the horizontal axis and absorbance as the vertical axis .
survival fraction ( sf ) was calculated as follows : sf ( % ) = ( od value of experimental group / od value of control group )  100% .
the experiment was repeated three times in parallel ; finally , time - survival curves were plotted according to the sf mean  standard deviation ( x-s ) of three samples .
cells were seeded in 24-well plates at 4  10 cells per well , two experimental groups were set up , and each group contained three parallel samples ; meanwhile , a control group was set up . after culturing for 24  h ,
each well was filled with 1  ml of medium containing optimal concentration of hyg and then cultured for 3  d and 5  d. streptavidin peroxidase ( sp ) method was used .
cells were fixed conventionally and stained , and the ones with appropriate morphological characteristics were selected under a 10x inverted microscope and photographed .
semiquantitative analysis of immunohistochemical images were performed using image j and image pro plus software . statistical processing : statistical processing was performed using spss software .
data were expressed as ( x-s ) , comparison among groups was performed by t - test , and correlation analysis was performed by paired chi - square test ; p < 0.05 was considered statistically significant .
cell survival curve describes the survival status of osteosarcoma cells at different doses and times ; see figure 1 .
as can be seen from figure 2 , the survival fraction of osteosarcoma hos cells decreases markedly with the passage of time , and no significant late stage cell proliferation is present .
correlation analysis showed that the cell survival fraction was negatively correlated with the drug dose ( p < 0.01 ) .
the morphological changes of osteosarcoma , after hyg treatment , and untreated osteosarcoma cells , under light microscopy , form as shown in figure 3 .
immunohistochemical images were analyzed using image pro plus software ; the results showed a gradual increase in gray scale value of each image , which indicated gradual decrease in protein content of each image .
the data showed that the htert and bcl-2 protein expressions in the experimental groups were apparently lower than the control group and that the expression intensity was negatively correlated with time ( figure 4 and table 5 ) .
positive cell counting was performed on immunohistochemical images using image j software ; the higher the count of positive cells , the larger the number of cells expressing htert and bcl-2 protein , which also means wide distribution of htert and bcl-2 protein - expressing cells , and vice versa .
the difference was significant by paired chi - square test ( p < 0.05 ) ( table 6 ) .
the paired chi - square test found that htert and bcl-2 protein expressions have a high degree of consistency and a significant correlation ( p < 0.05 ) .
icariin is one of the main active ingredients in yinyanghuo ; pharmacological studies have shown that icariin has multiple pharmacological activities such as antitumor , immunity enhancement , cardiovascular function improvement , and endocrine regulation .
many tcm preparations with yinyanghuo as the monarch drug [ 6 , 7 ] used icariin as the investigation index ; therefore , the selection of icariin as the investigation index for screening of extraction process in this study is feasible and reasonable . 
 referring to the literature , the author also tried to select different mobile phases , acetonitrile - water , methanol - acetic acid , methanol - water - glacial acetic acid , and so forth , which resulted in poor separation effects at the position of icariin peak , with interferences from other small peaks ; when methanol-0.38% phosphoric acid solution ( 48  :  52 ) was used , icariin peak separated well from other impurity peaks , without interfering peaks . through the understanding of many behavioral relationships between htert and bcl-2 protein , this experiment aims to understand the effects of hyg on htert and bcl-2 proteins in osteosarcoma ( hos ) cells and possible correlation ; meanwhile , it provides a theoretical basis for clinical application of hyg .
typical osteosarcoma usually occurs in the 1020 age group ; nevertheless , it may occur in any age group , including infancy , childhood , and old age .
men have a higher incidence than women , with a male - to - female ratio of approximately 2  :  1 .
surgical treatment has greatly improved the prognosis of patients , but disability rate remains very high .
telomerase is expressed at a very high level in 90% of cancer cells , while it is rarely expressed in normal cells ; it is a recognized anticancer target .
htert is the rate - limiting enzyme of telomerase , which regulates the activity of telomerase   and has a parallel relationship with telomerase activity [ 14 , 15 ] . at present , many scholars have turned their eyes to htert , hoping to achieve a breakthrough for the treatment of cancer .
bcl-2 gene is the focus of everyone 's attention ; there have been many reports that bcl-2 protein can protect cells from apoptosis and increase the expression of htert [ 1719 ] , but there are also some experiments which demonstrated that bcl-2 protein is not correlated with , or even negatively correlated with , htert [ 20 , 21 ] .
the present study found that untreated osteosarcoma cells were mainly in short spindle shape under light microscope and were arranged tightly , with large nuclei , nuclear division was common , and cells were grown in multilayers when they were dense .
after treatment with hyg , cell morphology changed obviously , cells were mainly in polygonal shape and were arranged in a monolayer , cell size tended to be uniform , and nuclei were small and were deeply stained ; these results indicated the occurrence of morphological differentiation and changes .
osteosarcoma cell apoptosis was significantly increased with the increase of drug concentration ; cell apoptosis was also significantly increased with the extension of drug action time . under the effect of optimal concentration , with time as the axis ,
htert and bcl-2 protein expressions did not change significantly in the control group ; in the experimental group 3  d and experimental group 5  d , htert and bcl-2 protein expressions gradually weakened and were in a negative correlation with the time of drug action , which were consistent with relevant reports [ 22 , 23 ] ; the two were in a time - dependent relationship with the drug and showed a high degree of consistency with respect to expression .
the study results showed the presence of time dependence between hyg and htert but could not confirm that hyg was able to directly influence telomerase activity ; there existed high degree of consistency between bcl-2 protein and htert , but whether there exists mutual assistance between the two needs further research ; due to the discovery of telomerase activity in part of normal tissues , whether hyg has toxic side effects on them also needs further study ; there is also the possibility of false positive and false negative results in nonfull quantitative determination of htert and bcl-2 gene protein expressions ; tumor cells were in a low ph state , but the tumor cells in the present experiment were in a state where ph is 7.27.4 , which can not fully reflect the low ph state of malignant tumor tissues in vivo .
 to screen the optimal extraction process and content determination of active component of huangqi yinyanghuo group ( hyg ) and to study the effects of hyg on human telomerase reverse transcriptase ( htert ) and bcl-2 protein in osteosarcoma ( hos ) cells , providing the theoretical basis for clinical application of hyg in treatment of osteosarcoma , orthogonal design table l9(43 ) was used to design the extraction process of hyg , and icariin was taken as the investigation index to optimize the extraction process of hyg . 
 0.125 , 0.25 , 0.5 , 1 , 2 , 4 , and 8  mol / l hyg were taken to act separately on logarithmic growth phase osteosarcoma hos cells , cck-8 assay was used to determine cell viability , and immunohistochemical sp assay was used to determine the expression of htert and bcl-2 protein . 
 apoptosis rate was positively correlated with the dose of hyg , and the expressions of htert and bcl-2 protein were significantly decreased with the prolonged duration of action . under the effect of hyg , dose was negatively correlated with osteosarcoma cell survival fraction ; osteosarcoma cell survival fraction was positively correlated with htert and bcl-2 protein ; duration of action was negatively correlated with htert and bcl-2 protein ; and htert and bcl-2 protein were in a synchronous relationship . 
1. Introduction
2. Instruments and Drugs
3. Methods
4. Determination of Icariin Content
5. Results
6. Discussion