Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
2 Department of Pharmacology, Nanjing University of Chinese Medicine Hanlin College, Taizhou, China
Corresponding author details:
Liwei He, Ph.D
Department of Pharmacology
Nanjing University of Chinese Medicine Hanlin College, No. 6 Xueyuan Road
Copyright: © 2019 Huiqin X, et al., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The orthogonal test method was used to investigate the effects of solvent volume
fraction, extraction time and solvent on the extraction process. The results showed that the
best extraction process of the total lignans in Radix Isatidis is 70% ethanol, the extraction
time is 2 hrs, and the amount of solvent is 8 times. The inhibitory effect of the total lines of
Radix Isatidis on RSV was determined by MTT method and cytopathic effect (CPE) method.
The total lignans of Radix Isatidis significantly reduced the formation of plaques during
the phase of virus adsorption and virus replication. The plaques decreased slightly when
the virus entered the cell stage, indicating that the total lignans of Radix Isatidis may exert
their anti-RSV during the stage of virus adsorption and virus replication. The extraction
conditions of the Radix lignans obtained by orthogonal design experiments are stable and
Radix Isatidis; Total lignans; Orthogonal design; Antiviral
Radix Isatidis is a traditional Chinese medicine, derived from the dry roots of the
cruciferous plant Isatis indigotica. It has the effect of clearing heat and detoxifying cold
blood and pharynx [1,2]. Nowadays, Radix Isatidis is often used clinically for the treatment
of bacterial infections and viral diseases, including influenza virus, herpes simplex virus,
respiratory syncytial virus (RSV), mumps virus, hepatitis B virus, coxsackie virus and so
on [3-6]. It is commonly considered a broad-spectrum natural antiviral drug . However,
due to the complex chemical composition of Radix Isatidis, the discovery of the potential
medicinal components of Radix Isatidis and the mechanism of action is seriously restricted
. The view that there may be a synergistic effect of Banlangen on the human body has
been reported. The theory of “pharmacy superposition” proposed by Professor Cai  of
Peking University shows that most of the active ingredients with similar active structures
act on one or more similar targets. Different types of compounds have been isolated from
Radix isatidis including indirubin, clemastanin B, alkaloids, lignans, and flavonoids [10,11].
The chemical composition of Banlangen mainly includes three major categories: alkaloids,
lignans and organic acids. At present, studies on Banlangen focus on the study of total
alkaloids and total organic acid sites [12-14], and there are few reports on the study of
lignans. However, the antiviral effect of lignans is significant [15,16], and the total lignans
in Radix Isatidis occupies a larger proportion, so the antiviral effect of total lignans in
Banlangen cannot be ignored. The extraction process of total lignans from Radix Isatidis
was optimized, and its antiviral effect was studied. It provides a theoretical and practical
basis for the effective utilization of Radix Isatidis medicinal resources and the synergistic
antiviral effects of Radix Isatidis.
Cells and viruses
Human laryngeal carcinoma epithelial cells (Hep2 cells) were kindly provided by the Institute of Pediatrics, Nanjing University of Chinese Medicine; RSV type, a long strain was provided by Wuhan Institute of Virology, Chinese Academy of Sciences.
Drugs and reagents
Banlangen (Origin: Bozhou, Anhui Province, batch number:20171211), medicinal
herbs were provided by the market of Anhui Bozhou medicinal herbs, were identified to
meet the “People’s Republic of China Pharmacopoeia” (2015 edition) standards; sodium
hydroxide (batch number:150708); hydrochloric acid (batch number: 20141126); Methanol (analytical alcohol, batch number: 20170110); Ethanol
(batch number: 20161216); Ribavirin (Shanghai Yuanye Bio, Batch
No. 36791045); DMEM high-glucose medium (US HyClone, Lot No.
AAK207235); Pancreatin , Fetal calf serum (US GIBCO, Lot 1801539,
20171026); MTT, DMSO, BSA (Sigma USA, batch number C10036912,
045M4080V, 10735078001); RIPA, phenylmethylsulfonyl fluoride,
BCA kit, loading Buffer (Shanghai Biyuntian Biological Co., Ltd.,
batch number is 0123251105, 0421323721, 03161712517,
0140260519);Phosphatase inhibitor, protease inhibitor (Jiangsu
Jikai Biological Technology Co., Ltd., batch number KGP602180214,
KGP603024523); anti-GAPDH antibody, goat anti-rabbit fluorescent
secondary antibody; Protein Marker (Runsheng Biological Co., Ltd.,
batch number 03018120); RSV F antibody (batch number sc-101362,
purchased from Santa Cruz Biotechnology); RSV G antibody (batch
ab94966, purchased from Santa Cruz Biotechnology); 1:29 bis-Acr,
10% SDS, APS (US GIBCO, Lot No. B0153K0103185, 3642A462,
B016M052548); TRIS, Glycine (Beijing Soley Company, Lot 562B018,
803B0156); Skim milk powder (Shanghai Wulian Technology Co.,
Ltd., Lot No. 1216542).
Optimization of total lignan extraction process of Radix Isatidis
Using L9 (34) orthogonal design [17,18] (Table 1), the volume
fraction, solvent amount and extraction time of ethanol were
investigated, and the extraction rate and total lignans content
obtained under various conditions were determined. The total lignans amount was measured by ultraviolet spectrophotometry.
Using larch resin alcohol as a reference substance, through fullwavelength scanning, it can be seen that the reference substance
and test solution have the same maximum absorption wavelength at
280 nm, so it is determined to measure at 280 nm. Pipette precision
pipette a certain volume of larch resin alcohol standard solution,
place it in a 10 ml volumetric flask, add methanol to dilute to volume,
and shake, to obtain concentrations of 0.01 mg/ml, 0.02 mg/ml, 0.04
mg/ml, 0.08 mg/ml, 0.16 mg/ml, 0.32 mg/ml standard solution,
respectively. The methanol solution was used as a blank solution to
determine the absorbance at the maximum absorption wavelength of
the above prepared standard solutions with different concentrations.
The concentration of the standard solution was plotted on the
abscissa, and the absorbance value A was plotted on the ordinate to
draw a standard curve. The total lignans content was calculated and
the results are shown in (Table 2).
|Level||A ethanol volume
Table 1: Extraction process factor level
|Test sequence||A ethanol
fraction/% ||B extraction
Table 2: Orthogonal test scheme and results of extraction process
Orthogonal test results show that the total rate of lignans in
Radix Isatidis decreases with increasing ethanol volume fraction.
The Table 3 variance analysis showed that the three factors of
ethanol concentration, solvent doubling amount and extraction time
had significant effects on the total lignan content of Radix Isatidis.
According to the extreme difference of the Extraction rate, the
effects of various factors on the rate of total lignans were as follows:
ethanol volume fraction> extraction time> solvent amount. The
pharmacological effect was determined by the total lignans content
of Radix Isatidis. Therefore, the total lignan content is the main
indicator in this experiment. Because the amount of solvent has little
effect on the experimental results, adhering to the principle of saving
resources, 8 times the amount of solvent is selected for extraction.
Therefore, the preferred process conditions are 70% ethanol volume
fraction, 2 h extraction time, and 8 times solvent.
|Factors||Sum of squares
Table 3: Analysis of variance
Macroporous resin enrichment and purification
The total lignan content of Radix Isatidis was extracted according to the preferred process conditions as described above, the solvent was recovered in a low pressure environment, and the sample was prepared to have a mass concentration of 1 g/ml, adjusted to pH=7 with NaOH, and the macroporous resin [19,20] was reprocessed and then wet-packed. Column, the sample was applied to the column, statically adsorbed for 12 hours, and eluted with distilled water to remove unadsorbed components and water-soluble impurities, and then eluted with different volume fractions of ethanol solution, identified by ultraviolet spectrophotometry, and identified as the eluent of the lignan component was dried under reduced pressure to obtain the total lignan of Radix isatidis.
Determination of total lignan content
Precision weighed total lignan sample 0.6 mg, placed in a 10 ml volumetric flask, diluted with methanol to determine the volume, that is, the sample solution. The total lignan content was determined under the same conditions as described above. The total lignan content in the sample after isolation and purification was 74.87%. Total lignan determination methodological investigation.
1 ml of the test solution was taken and measured as described above (n=6), and the result showed that the RSD value was 0.235%. It shows that the precision of the instrument is good.
Six samples (n=6) of the total lignan sample of Radix Isatidis were taken in parallel and measured as described above. The results showed that the RSD value was 2.03%, indicating that the method has good reproducibility.
Take the appropriate amount of total lignans from Radix Isatidis, dissolve with methanol, and make up to volume. Take 1 ml of the test solution, and measure the absorbance at the time of 0 min, 5 min, 15 min, 30 min, 1 h and 2 h, the absorbance was 0.2305, 0.2386, 0.2317, 0.2400, 0.2339 and 0.1538, respectively. It indicates that the sample solution has good stability within 1 h.
The sample was accurately weighed, and an appropriate amount
of the reference substance was added and measured according to the
above method. The recovery rates of the samples were calculated
to be 98.59%, 103.15%, 98.76%, 99.53%, 102.58%, and 98.36%,
Figure 1: The effect of lignans of Radix Isatidis on Hep2 cells
survival rate (x ± s, n=6)
Figure 2: The effect of Ribavirin on Hep2 cell survival rate(x ± s, n=6)
Direct anti-RSV study of total lignans
Hep2 cells were seeded in 6-well plates, and different
concentrations (50, 100, 200 μg/mL) of total lignans were incubated
with RSV on ice. The whole process is carried out on ice to prevent
the virus from entering the cell. After 2 h of incubation, the cells were
washed with pre-cooled medium to remove unbound virus. The cells
were then lysed and the RSV G and F proteins used to detect cell
attachment were analyzed by immunoblot . The result is shown
in Figure 3.
Figure 3: Lignans treatment suppresses RSV F and G protein expression
Hep2 cells were seeded in 6-well plates and incubated with RSV
(500 μL virus titer of 100 TCID50/well) for 2 hours on ice, allowing
virus attachment in the presence or absence of the indicated
concentration of drug. The non-attached virus was removed with icecold medium and then allowed to adsorb at 37°C for 2 hours to allow
the attached RSV to enter the host cell. At the end of the incubation
period, the culture medium was removed and the culture was replaced
with agarose containing 1% of 2.5% calf serum and incubated at 37°C
for 6-7 days. The formation of plaques was observed daily. The result
is shown in Figure 4.
Figure 4: Inhibitory effect of lignans on RSV infection
Hep2 cells were inoculated into 6-well plates, 500 ul of 100 TCID50 virus solution was added to each well, and placed in a constant temperature incubator for 2 h, shaking once every 20 min. After the end of the adsorption, the virus suspension in the culture plate was discarded, and replaced with agarose containing 2.5% calf serum 1%, and different concentrations of the drug were added, and the mixture was incubated at 37°C to observe the formation of plaques every day. The result is shown in Figure 4.
The RSV F protein and RSV G protein were not expressed in
the normal cell group, and the RSV F protein and RSV G protein
expression levels were higher in the virus group, indicating that
the virus adsorption amount was higher in 2 hours. Compared with
the virus group, each drug group RSV The expression of F protein
and RSV G protein decreased to varying degrees (p<0.01). With the
increase of drug concentration, the expression of RSV F protein and
RSV G protein decreased in turn.
The chemical composition of Radix Isatidis is very complicated, which seriously hinders the research process of Radix Isatidis. It has been shown in the literature that compounds with larger content may play a leading role in the overall content and efficacy of such components , and the total lignan content has a large proportion in Banlangen, and its antiviral effect is diverse and the mechanism of action is not clear. The study on the antiviral effect of total lignans is of progressive significance to elucidate the antiviral mechanism of Radix Isatidis, so its antiviral effect needs to be proved. However, since the lignans are mainly polar substances and there is no specific color reaction and identification method, it is difficult to enrich, because the research in this part is still blank, so the extraction and purification of the total lignans from Radix Isatidis the process needs to be unified.
In this study, we used orthogonal test to optimize the extraction process of total lignans from Radix Isatidis. The effects of ethanol concentration, solvent dosage, and extraction time on the total lignans content of Isatis indica were investigated. The method is stable, reliable and suitable for applicability; macroporous resin adsorption separation technique was used to separate and enrich the extracted samples. For the problem of how to effectively avoid the absorption of impurities, our study uses the wavelength, which the total lignans have higher UV absorption and the impurity has no or less absorption, as the detection wavelength of this experiment to avoid the interference of impurities. For the problem of how to purify the total lignans of Radix Isatidis more effectively, this study fully considers that the research object is the headquarters with the same basic nuclear structure, according to the principle of similarity of ultraviolet absorption of the same basic nuclear structure, so Ultraviolet spectrophotometry was used to collect the lignans components in sections, and other components of the UV absorption spectrum were discarded to purify and increase the total lignan content, which provides a basis and guarantee for the study of the pharmacological effects of the total lignans in Radix Isatidis.
For the study of the pharmacological activity of the total lignans
in Radix Isatidis, we first separated the total lignans from Radix
isatidis and used plaque reduction experiments (CPE method is a
classic method for studying viral infection experiments. This method
is simple, intuitively), the anti-RSV effects were studied from the
three stages of adsorption, entry and replication of virus invading
cells. The results showed that the total lignans of Radix Isatidis could
significantly inhibit the formation of plaques during RSV adsorption
and replication. Although this study shows that the total lignans of
Radix Isatidis can exert antiviral effects during the adsorption and
replication stages, it is still unknown for the mechanism of how the
target exerts antiviral activity. And because this article is limited to
respiratory syncytial virus, it does not mean that the total lignans are
effective against all viruses. So, further research is needed.
This project was supported by National Natural Science
Foundation of China (No. 81473316), Natural Science Foundation of
Jiangsu Province (No.BK20151356 and BK20161320). The project
has been supported by the Jiangsu Provincial Graduate Research and
Innovation Project (No. KYCX18_1586).
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