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22Study on Aloe vera polysaccharides with anti-HSV-1 activity
Yanxin LIU, Yanping SUN, Qiuhong WANG, Zhibin WANG, Haixue KUANG*
Key Laboratory of Chinese Materia Medica, Heilongjiang University of Chinese Medicine, Harbin,
China.
Abstract:Objective: The aim of this study is to research the relationship between the effects of Aloe vera polysaccharides (AP) with different molecular amounts on the anti-Herpes simplex virus type 1 (HSV-1) activity and to find out the optimal molecular amount on the anti-HSV-1 effect. Methods: Four Aloe vera polysaccharides AP-1, AP-2, AP-3 and AP-4 were isolated and purified from Aloe vera, and their molecular weight was determined by HPSEC. By using MTT colorimetric method, the cytotoxicity of each AP sample on Vero cells was measured, and inhibition effects of AP samples on HSV-1 were studied by in vitro cell culture assay. As the evaluation index includes the half toxic concentration (CC50), cytopathic effect (CPE), half inhibitory concentration (IC50) and therapeutic index (TI), the antiviral effect of AP samples were compared. Results: Vero cell toxicity of all the AP samples was low, with CC50 higher than 5 000 mgL"1. Four AP samples showed different degrees of anti-HSV-1 activity, and the antiviral activity enhanced with the dose increased within a certain concentration range. Among the four samples, AP-3 showed good antiviral activity with IC50 of 6.3 mg-L"1 and TI higher than 793.6. Conclusion: AP-3 has a good inhibitory activity on HSV-1, indicating that 80 kDa may be the best molecular weight against HSV-1.
Key words: molecular weight; Aloe polysaccharides; antiviral; HSV-1
Aloe vera is a liliaceous perennial evergreen succulent herbs of the genus Aloe[1], rich in a variety of active ingredients, enhance immunity, promote wound healing, antiviral, anticancer, antibacterial, anti peptic ulcer, resistance to radiation damage, diabetes and other function[2]. In recent years, studies have shown that polysaccharide of monosaccharide natural polymer, its properties and pharmacological effects were closely related and its molecular size, different molecular weight or structure of the polysaccharide may have different pharmacological activities.
Therefore, this study through the investigation of different molecular weight AP active relationship with its anti HSV-1, in order to further clarify the AP effective parts and in antiviral resistance to HSV-1 provides the reference for the development and utilization of medical field.
1. Materials and methods
1.1 Materials
Aloe vera whole leaf producing (100:1) (hainan nine blessing to science and technology development co., LTD.); Acyclovir (acyclovir ACV, listed the joint pharmaceutical co., LTD. Harbin, batch number is 120603). African green monkey kidney cells Vero cells (ATCC, USA); Herpes simplex virus HSV - 1 (F strains, the ATCC VR733, provided by the immunology lab of Harbin medical university); According to the conventional method[3] culture cells reproduce viruses, HSV - 1 half of the infected quantity TCID50 virus for 10 - 52/100^L.
1.2 Methods
1.2.1 AP preparation of different molecular weight
Aloe vera whole leaf producing, extracted by of 95% ethanol at room temperature 3 times, vacuum suction filter to get liquid to dissolve with distilled water after drying, put in anhydrous ethanol, in turn, make its final concentration of 40% and 80%, and each time the centrifugal separation, precipitation, suction filtration, vacuum drying, get two polysaccharide components. Respectively on Amberlite again FPA-90Cl" column, and tandem Amberlite IRC-84+ column, in order to take off in the coarse polysaccharide protein,and then by DEAE Cellulose-DE-52 and Sephacryl gel chromatography column further purification, freeze drying, AP-1, AP-2, AP-3fPAP-4 Its high gel filtration chromatography (HPSEC) map display for the single peak symmetry (as shown in Figure 1), showed that more than four are of uniform molecular weight polysaccharides or
107
polysaccharide. Measured by HPSEC-ELSD, AP-1, AP-2, AP-3fPAP-4 relative molecular weight were 451.3 kDa, 1221.8 kDa, 80.2 kDa and 3.6 kDa. Isolated from the samples of the AP with distilled water dissolved into 10 g/L, after the high pressure steam sterilization, at 4 °C in freezer for later use.
0 -|—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i 0 H—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i 0 2 4 6 S 10 12 14 16 1820222426 28 30 3234 0 2 4 6 8 10 12 14 16 1820 22 24 26 28 30 3234
mill mill
Figure 1. Four AP high gel filtration chromatography (HPSEC) map
1.2.2 AP period of the antiviral activity of different molecular weight
1) AP the determination of cytotoxicity
Vero cells administered in 96-well culture plate, after waiting for the grown monolayer cells, refuse to go to cleaning fluid, respectively to join AP-1,AP-2,AP-3fPAP-4water dilution of seven to times diluted solution(4 000.0, 2 000.0, 1 000.0, 500.0, 250.0, 125.0, 62.5 mgL-1), the concentration of each 4 holes, each hole 100^L, at the same time a negative control group (with 100 ^L DMEM maintain fluid), positive control group (ACV for times diluted to join listed). Continue to cultivate 72 h at 37 °C 5% CO2, calculation of cell survival rate, the AP sample half poisoning of cell Concentration (Cytotoxic Concentration, referred to as CC5o, mgL-1) and the maximum non-toxic Concentration (Maximal Non - Cytotoxic Concentration, referred to as MNCC, mgL-1). Cell survival rate = bore samples A/control A*100%
2) AP in vitro antiviral activity assay
Vero cells are grown in 96-well plates to grown monolayer, abandon to sustain liquid, will each AP solution under test with the highest concentration of not more than MNCC times diluted, horizontal inoculation in sequence in each hole, 50^L per hole, then add 50^L contains 100 TCID50 virus suspension, HSV - 1 each sample concentration after four holes, at the same time a negative control group (with 100^L DMEM maintain liquid), the virus control group (50^L TCID50 virus suspension and 50^L DMEM maintain fluid) and positive control group (50^L to 100 times dilution of ACV and 50pL 100TCID50 virus suspension of HSV - 1). Put 96-well plates at 37°C and 5% CO2 incubator in training, daily status of cell growth and form. When the virus control group ++++, and the negative control group, normal record holes each of type A virus inhibition rate, mark is calculated values and CPE (" - "said for CPE occurred; "±"said 10% the following CPE;" + "says
less than 25% in CPE;" + + "said 26% ~ 50% of CPE;" +++ "said 51% ~ 75% of CPE;" ++++ "said happened CPE of 76% ~ 100%), and the AP sample half inhibitory concentration (50% inhibited concentration, IC50), and calculate the therapeutic index (treatment index, TI = CC50 / IC50). Among them, the virus inhibition rate = (experimental groupA- virus control group A)/(negative control groupA- virus control group A) x100% 3) Statistical processing
Using SPSS 13.0 for experimental data processing, t test, represented by x±sa P < 0.05 have statistical significance. 2. Results
2.1 The AP on cell toxicity test
Table 1 Samples of different concentrations of AP's influence on Vero cell survival( x±s, n=5)
MNCC /mgL-1
Cell survival rate /%
samples 4000.0 2000.0 1000.0 500.0 250.0 125.0 62.5
/mgL-1 /mgL-1 /mgL-1 /mgL-1 /mgL-1 /mgL-1 /mgL-1
AP-1 62.0±2.22) 86.2±1.32) 113.7±4.81) 114.2±4.6 109.6±3.5 105.3±3.1 112.2±4
AP-2 58.4±2.82) 77.3±2.22) 103.2±3.11) 108.6±3.4 101.0±4.4 113.1±4.2 109.9±3
AP-3 82.5±1.12) 90.5±4.82) 107.5±5.81) 103.4±2.7 114.1±2.9 107.2±3.1 118.4±3
AP-4 67.8±2.02) 82.4±3.52) 112.7±4.71) 104.1±4.9 115.3±4.3 102.5±5.3 115.0±4
ACV
CC50 /mgL-
>5
000.0
>5
000.0
>5
000.0
>5
000.0
600.0
1 000.0
1 000.0
1 000.0
1 000.0
Note: 1) compared with negative control group (DMEM) P < 0.05; 2) P < 0.01). 2.2 Comparison of different molecular weight AP antiviral activity
Table 2 the AP sample for CPE caused by HSV-1, virus inhibition rate, and IC50 values of TI
(n=5)_i_
C/mgL-
samples Evaluation method 1 000.0 500.0 250.0 125.0 63.0 31.0 16.0 8.0 4.0 1C50 /mgL-1 TI
AP-1 CPE + + + + ++ ++ +++ +++ ++++
Virus inhibition 86.6 85.2 84.6 82.6 74.2 52.2 34.9 26.3 20.8 26.1 >191.6
rate/%
AP-2 CPE ++ ++ ++ ++ +++ +++ +++ +++ ++++
Virus inhibition 72.5 69.1 63.0 57.8 48.4 41.5 34.3 25.6 23.4 71.9 >69.5
rate/%
AP-3 CPE ± ± + + + ++ ++ ++ +++
Virus inhibition 95.0 92.3 85.0 81.8 76.1 67.4 62.3 58.1 42.3 6.3 >793.6
rate/%
AP-4 CPE + + ++ ++ ++ ++ +++ +++ ++++
Virus inhibition 88.9 82.3 74.9 72.3 71.6 70.4 49.8 37.1 12.7 24.5 >204.1
rate/%
ACV CPE ± ± ± + + + + ++ ++
Virus inhibition 97.3 95.2 92.7 87.4 85.0 82.1 78.9 74.6 66.2 0.9 >645.2
rate/%
Note: "±" shows less than 10% of the CPE occurred; "+" shows less than 25% of the CPE occurred; "++" shows 26% ~ 50% of the CPE occurred;"+++" shows 51% ~ 75% of the CPE occurred;"++++" shows 76% ~ 100% of the CPE occurred
3. Disscusion
Polysaccharide has many kinds of biological activity, because of the source of the polysaccharide is widespread, even the same sources of plant polysaccharide molecular structure and molecular weight of each are not identical. Different molecular weight polysaccharides may also have different pharmacological activities. Similarly, polysaccharide antiviral activity is not only related to its molecular composition, but also has a lot to do with its relative molecular mass. Combining with the physical and chemical properties of the antiviral activity of polysaccharides studied, aloe polysaccharide after purification from 4 kinds of uniform molecular weight polysaccharides, AP-1,AP-2,AP-3fPAP-4. You can see by the liquid chromatogram, polysaccharide, AP-1,AP-2,AP-3fPAP-4 are the single peak symmetry, to prove that,AP-1,AP-2,AP-3fPAP-4 have is pure single components. This experiment focuses on antiviral activity of the four AP filtering, using in vitro cell culture method, studies the four kinds of AP samples for CPE inhibition caused by HSV - 1, find the AP sample CPE caused by infection of HSV-1 all have certain inhibition. The AP-3 anti HSV-1 the best effect, IC50 of 6.3 mgL-1, TI is greater than 793.6, the molecular weight of polysaccharides was 80.2 kDa. In addition, the result shows that the size of the order four AP inhibit HSV - 1 for: AP-3 > AP-4 > AP-1 > AP-2; Four AP treatment index size order: AP-3 > AP-4 > AP-1 > AP-2. Small molecular weight AP antiviral effect is good in the antiviral effect of molecular weight.
But when the molecular weight is less than 80 kDa, antiviral effect of small molecular weight is less. In theory, it is generally believed that the activity of polysaccharide is related to molecular weight, polysaccharide, the bigger the molecular weight, the greater the molecular volume, is not conducive to enter the body across a membrane biological activities, relatively small molecular weight polysaccharides is easier to combined with the active site, instead the relative molecular weight is too low, can't form the activity of the aggregation structure. Therefore, relative molecular weight of the appropriate size of polysaccharide, the active is high. Thus preliminary inference, the molecular weight is probably around 80 kDa AP antiviral action of the optimum molecular weight. With ACV resistant HSV-1 medicine listed in comparison, due to natural AP, AP - 3 for its CC50 obviously than ACV is much higher, the listed with high efficiency, low toxicity resistant HSV-1 concurrently at the same time, this for further research on the antiviral activity of AP, AP of biological activity and structure-activity relationship of in-depth study provides the basis for the development of new drugs in anti HSV-1, has the very vital significance. So far, this experiment is to AP function at the beginning of the deep, the AP anti HSV-1 mechanism still is not clear, therefore, for AP mechanism of HSV-1, we will continue to in-depth study.
AP-3 has good activity, inhibit HSV-1 tip of molecular weight 80 kDa role may be resistant HSV-1 the best molecular weight.
Reference
1. Liang Z.Y., SunD.Y., Zhang J.Y., et al. Aloe vera skin antioxidant activity research. Lishizhen Medicine and Materia Medica Research. - 2011 May. -22 (5). -P. 1152-1153
2. Chang X.L.,Feng Y.M., Cong L.H., et al. Review update on bioactive functions of aloe. Food Science and Technology. - 2007 May. -32 (5). -P. 10-13
3. E Z. Tissue culture and molecular biology technology. Beijing: Beijing publishing house. - 1995
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