A new biocompatible microemulsion increases extraction yield and bioavailability of Andrographis paniculata

Chinese Journal of Natural Medicines 2016, 14(9): 0683−0691

Chinese Journal of Natural Medicines

A new biocompatible microemulsion increases extraction yield and bioavailability of Andrographis paniculata LIU Xiao-Yan1, NIU Xin2, FENG Qian-Jin3, YANG Xue-Zhi2, WANG Dan-Wei2, ZHAO Tong1, LI Lei1, DU Hong1* 1

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China; School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing 100029, China; 3 Shanxi University of Traditional Chinese Medicine, Taiyuan 030024, China 2

Available online 20 Sep., 2016 [ABSTRACT] The purpose of this study was to design and prepare a biocompatible microemulsion of Andrographis paniculata (BMAP) containing both fat-soluble and water-soluble constituents. We determined the contents of active constituents of BMAP and evaluated its bioavailability. The biocompatible microemulsion (BM), containing lecithin and bile salts, was optimized in the present study, showing a good physical stability. The mean droplet size was 19.12 nm, and the average polydispersity index (PDI) was 0.153. The contents of andrographolide and dehydroandrographolide in BMAP, as determined by high performance liquid chromatography (HPLC), were higher than that in ethanol extraction. The pharmacokinetic results of BMAP showed that the AUC0-7 and AUC0→∞ values of BMAP were 2.267 and 27.156 μg·mL−1·h‒1, respectively, and were about 1.41-fold and 6.30-fold greater than that of ethanol extraction, respectively. These results demonstrated that the bioavailability of and rographolide extracted by BMAP was significantly higher than that extracted by ethanol. In conclusion, the BMAP preparation displayed ann improved dose form for future clinical applications. [KEY WORDS] Andrographis paniculata; Biocompatible microemulsion; Andrographolide; Bioavailability; Ethanol extract

[CLC Number] R969.1

[Document code] A

[Article ID] 2095-6975(2016)09-0683-09

Introduction Andrographis paniculata (Burm.f.) nees, a famous Chinese medical plant, belongs to the family of Acanthaceae and contains a variety of bioactive ingredients, such as andrographolide, neo-andrographolide, deoxyandrographolide, and methoxy flavonoids [1]. According to the traditional Chinese medicine (TCM) theory, Andrographis paniculata (AP) can clear heat and toxicants, cool blood, and diminish swelling [2]. The major active constituents have positive effects on regulation of immunity [3] and anti-inflammatory [4], antibacterial, and anti-

[Received on] 17- Apr.-2015 [Research funding] The work was supported by the National Nature Science Foundation of China (No. 81102815) and New Teacher Fund for Doctor Station, the Ministry of Education of China (No. 20110013120017). [*Corresponding author] Tel: 86-10-84738616; Fax: 86-1084738616; E-mail: [email protected], [email protected] These authors have no conflict of interest to declare. Copyright © 2016, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved

oxidant activities [5-6], making AP “natural antibiotics” and conferring high value in treatment of infectious [7-8], cardiovascular and cerebrovascular [9-10], digestive and other diseases [11-12]. In addition, these active constituents have also been studied for treatment of cancer [13], lung injury and mental illnesses [14-15]. Water, ethanol, and alkali extraction and acid precipitation are frequently used to extract active ingredients of AP for medicinal purposes at present, but these methods have certain drawbacks. Some components in AP can be extracted by water, but the extraction yield for andrographolide is poor. The yield of andrographolide is high with ethanol extraction, and ethanol in moderate concentration is also suitable to extract flavonoid glycosides, but has some defects like time-consuming, use of large volume of solvents and lower yield, resulting in waste of resources. The ring-opening derivatives of andrographolide are soluble in water with alkali extraction method, so a better extraction yield of andrographolide and dehydroandrographolide can be obtained, but the extraction rate of total flavonoids in an alkaline environment is significantly decreased at the same time [16-18]. Microwave-assisted extraction (MAE) [19], decompressing inner ebullition (DIE) [20], and ultrasonic extraction (UE) [21] have

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been used to extract the active ingredients in AP, but the drawback is that andrographolide and flavonoids cannot be extracted simultaneously by these methods. Additionally, high-speed counter current technology (HSCCC) [22], supercritical CO2 extraction technology (SFE) [23] and other environmental microextraction (ME) [24] processes have not been widely used in recent years, because of expensive or stringent conditions. Some improved traditional extraction technologies can also improve the extraction yield of traditional Chinese medicines [25-26], but whether these methods could solubilize all of the active ingredients with different polarities in AP remains elusive. Therefore, finding proper solvents to simultaneously solubilize the components with various polarities is the key to extract active ingredients of AP comprehensively and effectively. Microemulsion is a transparent or translucent isotropic colloidal system that spontaneously forms from the combination of water, oil, surfactant and co-surfactant in a proper proportion. As a thermodynamically stable system, it can be used as pharmaceutical carrier to solubilize different polarity drugs [27-28], which may be suitable for extracting all the active ingredients of AP. In applications of extracting TCM, studies have shown that extraction time could be reduced and protein activity could be maintained when extracted by microemulsion [29-31]. We have used microemulsion as a drug carrier to contain both water- soluble and fat-soluble components in Angelica and generated a new Angelica preparation with better efficacy and bioactivity [32]. In general, biocompatibility refers to the response capacity of biomaterials in dynamic and static changing process in vivo; a good biocompatibility means that the preparation is relatively stable in biological system, without any effects on clinical responses and/or toxicity of the main drugs [33]. Biocompatible microemulsion (BM) has various characteristics of biocompatibility that the four phases (water, oil, surfactant, and co-surfactant) are harmless to the organism and have no rejection reaction at the same time. Cholesterol is a derivative of cyclopentanoperhydrophenanthrene, which is similar to the fat regarding solubility and suitable for forming oil/water (O/W) microemulsion. As an essential component of cell membrane, with low toxicity, this compound is very suitable for preparing drug carrier system. Lecithin has strong surface and physiological activity, because of the fatty acid structure with strong lipophilic and the amino and phospholipid structures with strong hydrophilic of phosphatidylcholine (PC). The main component of Lecithin, a natural surfactant, has been provided a strong foundation in emulsifying ability by this amphoteric compound [34]. Bile salts (BS) have strong solubility for many drugs [35] and can accommodate more water-insoluble drug molecules after adding PC. The molar ratio of lecithin and bile salts is 1 : 3 (the physiological proportion of human bile) [36-38]; when lecithin is mixed with hydrogenated castor oil (RH-40) with the ratio of 1 : 1, the solubiliz-

ing power could be enhanced significantly. The aim of the present study was to prepare a microemulsion with optimal formulation using mixed experimental design (a method of mixing a number of different materials into a stable substance), and use the microemulsion to prepare BMAP, in order to establish a new, more efficient and suitable extraction method that could overcome the inadequacies of the current AP preparations.

Materials and Methods Materials AP was purchased from Beijing Huamiao Traditional Chinese Pharmaceutical Engineering and Technology Developing Center (Beijing, China), identified as the dried aerial parts of Andrographis paniculata (Burm.f.) nees, and stored in dark hermetically; andrographolide and dehydroandrographolide were purchased from National Institute for Food and Drug Control (Beijing, China); PC50 lecithin was purchased from Shanghai Taiwei Pharmaceutical Co. (Shanghai, China); cholesterol and isopropyl myristate were purchased from Sigma-Aldich Co. (Shanghai, China); pig bile salts was purchased from Beijing Shuangxuan Microbiological Media Products Factory (Beijing, China); polyoxyethylene hydrogenated castor oil was purchased from BASF Co. (Guangzhou, China); acetonitrile and methanol were chromatographically pure, which were purchased from Tianjin Yongda Chemical Agent Development Center (Tianjin, China); and all other chemicals and reagents were of analytical pure and used without further purification. Animals Twelve (6 for each sex) Japanese big-eared rabbits (Oryctolagus cuniculus) were obtained from Vital River Laboratories (Beijing, China) and housed under sterile condition with free access to common diet and tap water for 2 weeks before experiments. The relevant experiment protocols were approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. Preparation of BM We chose cholesterol and IPM as oil phase, bile salts/phosphatidylcholine as a surfactant, and RH-40 and ethanol as co-surfactants for preparing BM. Design Expert 17.0 was used for experimental design, as the testing of regression equation, the determination of the constant value and the most value, as well as other functions were integrated by this software. D-optimal design in mixture designs was selected to optimize the formulation of BM (Table 1). The final BM was prepared according to the optimized formula. First, a certain amount of lecithin was dissolved in RH-40 to make a uniform mixture, to which the mixed solution of cholesterol and IPM (proportional to the amount of 95% ethanol in the optimized formula) was added with stirring evenly. The BS dissolved in double distilled water was added into the afore-

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mentioned mixture slowly with continuous stirring by using a constant temperature magnetic stirrer until a transparent microemulsion was formed. After being appropriately diluted, the particle size and polydispersity index (PDI) of BM were measured by Malvern particle size analyzer (Malvern, Nano-zs, UK). The methods of regression analysis (backward) and cubic polynomial fitting were used to analyze the dynamic changes between the dependent variable (the particle size) and independent variables (all levels of the oil phase, aqueous phase, surfactant, and co-surfactant). Regression coefficients and constants were calculated before investigating the accuracy of regression equation with the indexes of fitting degree and the correlation coefficients and the optimal BM formulation was filtered by the method of analysis of variance. Determination of physicochemical property of the BM The droplet size of the BM was measured by Malvern particle size analyzer. The BM sample prepared according to the optimized formulation was appropriately diluted with double distilled water and filtered through a 0.45-μm microporous membrane in order to reduce the interference of particulate matter. The filtered BM sample was put into a standard quartz cuvette to measure the droplet with Malvern particle size analyzer, with wavelength of 658 nm, scan angle of 90º, and temperature of 25 ºC. The data were then analyzed by using the software package provided by the manufacturer. Each BM sample was measured for six times and the mean value was presented. The droplets of the BM were morphologically observed under a transmission electron microscope (TEM, JEM-1230, JEOL, Tokyo, Japan). The BM sample was diluted with double distilled water appropriately to avoid the aggregation of microparticles. When a liquid membrane formed directly, the BM sample was stained with 2% (W/W) phosphotungstic acid solution (pH 7) for 15 min and then dried at room temperature. An appropriate amount of BM sample was centrifuged twice (10 min each) at 1 000 g at room temperature, while the same BM samples without centrifugation were kept at 0, 4, 25, and 50 ºC for 3 months. Then the clarity and morphological changes of BM were observed by visual observation and absorbance detection at 500 nm wavelength using an ultraviolet spectrophotometer. BMAP preparation using different methods In our preliminary experiments, we compared extraction efficiencies of active ingredients in AP using BM or ethanol as extraction solvent. AP (20 g) was dissolved in 200 mL of BM, stirred evenly, and then extracted with ultrasonication for 30 min at room temperature, water bath for 30 min at 70 ºC, or cold macerating for 4 days at room temperature, respectively. The filtrates of BMAP extracted by these methods were collected after decompressed filtration, respectively. The procedures percolating extraction were as fol-

lows: 20 g of AP was dissolved in 20 mL of BM and stirred evenly, protected from light for 1 h to make AP wet or swell sufficiently, and then transferred to a percolator. After tiny air bubbles were eliminated, 180 mL of BM was added to allow for dipping for 36 h (sealed), and the filtrate of BMAP was collected in the process of percolate extraction at room temperature. Ethanol extract of AP (the concentration of ethanol was 28.8%) was prepared with the same extraction methods. Determination of andrographolide and dehydroandrographolide The reference solutions of andrographolide and dehydroandrographolide were prepared in methanol to form the final concentration of 0.1 mg·mL−1 separately. 2 mL of the BMAP or ethanol extract was dissolved in 6 mL of methanol, mixed with a vortex mixer for 120 s, and then centrifuged at 3 000 g for 10 min. The supernatants were filtered with 0.45-μm membrane filters and 20 μL was injected into HPLC system (Waters 2695/2996, USA) to quantify the contents of andrographolide and dehydroandrographolide. The HPLC system included a Waters 2690 equipped with a gradient controller, an automatic sample injector, and a 2996- photodiode array detector. Separation was performed on a C 18 column (150 mm × 4 mm, I.D. 5 μm, DIKMA, China). The mobile phase consisted of acetonitrile (A) and water (B) at a flow rate of 1.0 mL·min −1 with gradient elution as follows: 0−5 min, 22%−25% A; 5−30 min, 25%−30% A; and 30−50 min, 30%−40% A. The dual-wavelength was set at 225 and 254 nm, and the column was maintained at room temperature. Evaluation of bioavailability of the BMAP Japanese big-eared rabbits of 205−300 g (Vital River, China) were maintained in an air-conditioned animal quarter provided by the Laboratory Animal Center of Beijing University of Chinese Medicine (Beijing, China) at (22 ± 2) ºC and relative humidity of 55% ± 5%, with free access to water and diet. The animals were acclimatized for 5 days, fasted for 12 h, with free access to water, prior to experiment, and randomly allocated into two groups (6 in each group). The rabbits were anesthetized with 20% urethane and treated with carotid artery catheterization. About 5 mL of baseline blood sample was collected via the carotid artery and stored in heparin-anticoagulated tube until analysis. One group was administered ethanol extracts of AP while the other group was administered BMAP by gavage at a dose of 20 g·kg−1 (based on the calculated andrographolide concentration of 40 mg·kg−1). The medicated blood samples (5 mL) were collected into a heparinized tube via the carotid artery at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, and 7 h after drug administration. Following centrifugation at 600 g for 10 min at 4 ºC, plasma samples were obtained and stored at −20 ºC until analysis. An aliquot of plasma sample (100 μL) was mixed with 300 μL of absolute ethanol with a vortex mixer for 120 s and centrifuged at 1 000 g for 15 min. Then 20 μL of re-

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sulted solution was injected onto HPLC system for evaluating the oral bioavailability of the BMAP. Chromatography was performed using the method described above. Statistical analysis SPSS Version 20.0 for Windows (SPSS Inc. Chicago, IL, USA) was used for statistical analysis. The data are presented as means ± standard deviation (SD). Differences between groups were evaluated by post-hoc test and P < 0.05 was considered statistically significant.

Table 1 D-optimal design of mixture design of BM

Results and Discussion The formula of the BM After the type of surfactant, co-surfactant and the oil phase with the characteristics of good biocompatibility and biological activities were determined, the pseudo-ternary phase diagrams was used to screen the numerical boundary of the mixed surfactant, the oil phase and the aqueous phase for mixed design. The mixed surfactant (Smix) could be prepared when the Km values were 1 : 4, 1 : 2, 1 : 1, 2 : 1 and 4 : 1, and then mixed with oil phase, according to the volume proportion of 1 : 9, 2 : 8, 3 : 7, 4 : 6, 5 : 5, 6 : 4, 7 : 3, 8 : 2, and 9 : 1, which was performed in double distilled water with stirring by a magnetic stirrer until a clear transparent microemulsion was formed. The amount of added double distilled water was recorded in order to determine the microemulsion-forming boundary points. According to the titration results, the limit values of each phase with different Km were obtained as follows: the surfactant (X1): 0.060−0.300, co-surfactant (X2): 0.030−0.050, an oil phase (X3): 0.009−0.030 and the aqueous phase (X4): 0.620−0.901. Considering the impact of the ethanol content on solubilization effect, Km = 1 : 4 mixed experimental design (Table 1) was chosen as the optimized value because of the bigger microemulsion-forming area and the ability to form more stable microemulsion [39]. According to Table 1, a certain amount of surfactant and the oil phase were mixed and stirred by a magnetic stirrer before slowly adding the co-surfactant and water until a homogeneous BM was formed, and the BM was then diluted appropriately. The particle size and PDI were measured by Malvern particle size analyzer. The particle size was set as dependent variable versus the four independent variables. The methods of backward regression and cubic polynomial fitting were used to calculate the regression coefficients and constants whose accuracy was inspected by the fitting degree R2 and the correlation coefficient. The effect curve between the index and the factors was plotted according to the fitting equation (Fig. 1).

No.

A

B

C

D

1

0.238

0.034

0.013

0.715

2

0.290

0.040

0.020

0.650

3

0.300

0.030

0.009

0.661

4

0.118

0.044

0.018

0.820

5

0.291

0.050

0.009

0.650

6

0.179

0.039

0.009

0.773

7

0.165

0.050

0.030

0.755

8

0.290

0.030

0.030

0.650

9

0.300

0.030

0.009

0.661

10

0.223

0.044

0.023

0.710

11

0.060

0.050

0.009

0.881

12

0.179

0.030

0.018

0.773

13

0.060

0.040

0.030

0.870

14

0.061

0.030

0.009

0.900

15

0.270

0.050

0.030

0.650

16

0.060

0.050

0.009

0.881

17

0.118

0.034

0.013

0.835

18

0.060

0.040

0.030

0.870

19

0.291

0.050

0.009

0.650

20

0.290

0.030

0.030

0.650

Note: A: surfactant (0.060−0.300); B: co-surfactant (0.030− 0.050); C: oil phase (0.009−0.030); D: aqueous phase (0.620−0.901)

Fig. 1

Effect curve of BM in different proportions

An obvious trend of this effect curve is shown from the red region to the blue region (particle size changed from larger to smaller), indicating a great change in particle size that was impacted significantly by these factors. At the same time, the contour plot (the yellow area) projected by 3D shows that any change in the other two factors could be caused by any one of the three in the same contour line. Therefore, a significant interaction existed among surfactant, co-surfactant, and oil phase. Each index mentioned above was calculated by statistical analysis and the regression equation obtained as follows: Y = b0 + b1X1 + b2X2 + b3X3 + b4X4; The equations obtained by cubic polynomial fitting and the multivariate thrice multinomial obtained ultimately were

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as follows: Y = b0 + b1X1 + b2X2 + b3X3 + b4X4 + b5X1X2 + b6X1X3 + b7X1X4 + b8X2X3 + b9X2X4 + b10X3X4 + b11X1X2X3 Y = 19 503.724 06X1－2.591 65 × 106X2 ＋1.876 24 × 6

10 X3 －11 295.628 51X4＋ 2.506 63 × 106X1X2－3.153 74 × 106X1X3＋10 708.154 22X1X4－8.506 30 × 106X2X3 + 2.961 36 × 106X2X4 －1.385 06 × 106X3X4 ＋2.978 40 × 107X1X2X3。 In these formulas, Y is the measured value of the particle diameter, b0 is the intercept, b1−b9 are the regression coefficients and X1, X2, X3, X4 are the studied factors. The results of optimal formula and interactions between these factors and

indicators are shown in Table 2. The results indicated that the interaction among surfactant, co-surfactant and oil phase was highly significant (P < 0.000 1) and the regression equation could be used to estimate the values of each formula within the range of factors and levels. According to the minimum value of the equation, the minimum points (0.232, 0.031, and 0.013) were determined, and the optimal prescription of BM was surfactant: co-surfactant : oil phase : aqueous phase = 0.232 : 0.031 : 0.013 : 0.724. The BM was prepared under this optimum condition and the particle size and PDI of this BM were measured in order to investigate the physicochemical property.

Table 2 Variance analysis of optimal formula of BM Source

Sum of Squares

df

Model

1.10 × 10

7

10

Linear

65 162.97

3

40.22

1

Mean square

F Value

P-value

Prob > F

11 101.26

7

6.366 × 10

< 0.000 1

Sig.

21 720.99

6.366 × 107

< 0.000 1

40.22

6.366 × 107

< 0.000 1

7

Mixture AB AC

398.91

1

398.91

6.366 × 10

< 0.000 1

AD

42.56

1

42.56

6.366 × 107

< 0.000 1

7

< 0.000 1

BC

6 160.54

1

6 160.54

6.366 × 10

7

BD

42.44

1

42.44

6.366 × 10

< 0.000 1

CD

757.52

1

757.52

6.366 × 107

< 0.000 1

ABC

10 044.50

1

10 044.50

6.366 × 107

< 0.000 1

Pure Error

0.000

4

0.000

6.366 × 107

< 0.000 1

Cor Total

1.110 × 105

14

Physicochemical properties of the BM The BM prepared according to the optimized formula was a clear and transparent yellow-brown homogeneous system, belonging to O/W type. The stratification and flocculation did not appear when the sample was kept at room temperature for 24 h. The BM also remains the common status of microemulsion in appearance except the lighter color after being diluted with a small amount of double distilled water. But when the BM was diluted with water more than 3-fold, it flocculated and precipitated. The reason for that may be the big changes in the formula ratio destroyed the composition phase of the BM. The results showed that the BM we prepared was in line with the quality of a common microemulsion and could remain stable after appropriate dilution. The particle sizes of the BM measured by Malvern particle size analyzer for three times separately were as follows: 19.03, 19.23 and 19.20 nm and the PDI values were 0.165, 0.147, and 0.147, respectively. The appearance of the optimized BM was homogeneous spherical droplets, and the particle sizes were between 10−100 nm, which were in line with the characteristics of the common microemulsions (Fig. 2). The BM sample remained clear and transparent after

being centrifuged twice (10 min each time) at 1 000 g at room temperature. No change was found in the appearance and the OD values maintained stable when the BM samples were kept at 0, 4, 25, and 50 ºC for 3 months. The results indicated that routine changes in the environmental temperature may not affect the physicochemical properties of the BM. But the sample became turbid, flocculated and layered when kept at 80 ºC for 24 h, indicating that higher temperature may destroy the microstructure of the BM to a certain extent, which was associated with the thermodynamic properties of BM. Active ingredients of the BMAP The BMAP containing a variety of chemical compositions was obtained by using cold macerating extraction when BM was used as solvent and AP as extracting target. The diterpene lactones, the major compounds in AP, have a wide range of biological activities, such as inducing differentiation of cells [40], antihepatotoxic [41], improving bile secretion and changing its physical properties [42]. As the major diterpene lactones in AP, andrographolide and dehydroandrographolide have higher pharmacological activities than any other active ingredients of AP are used more in clinical practice; they were measured by HPLC in the present study (Fig. 3).

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Fig. 2 Particle size (nm) of the optimized BM measured by Malvern particle size analyzer and the micrograph of the optimized BM taken with the transmission electron microscope (× 40 000)

Fig. 3 The Contents of Andrographolide and Dehydroandrographolide extracted by Ultrasonic extraction, Cold macerating extraction, Water bath extraction, and Percolating extraction in BMAP and in Ethanol extract

As in Fig. 3, the contents of andrographolide and dehydroandrographolide in BMAP extracted by cold macerating extraction were higher than that extracted by other three methods. The contents of these two ingredients in BMAP were greater than that in ethanol extract, suggesting that BM was more suitable for extracting the diterpene lactones than ethanol and a higher extraction yield of andrographolide and dehydroandrographolide could be gained by cold macerating extraction. In our experiment, many small peaks indicating other chemical compositions in AP were found on the baseline from HPLC, suggesting that BM can extract more fat-soluble and water-soluble components at the same time and play the role of biological trapping effect better, compared with ethanol extraction (Fig. 4).

Pharmacokinetics of the BMAP The standard curve equation Y = 1.839 4 X − 0.306 8 (R2 = 0.992 4) was calculated by linear regression over the range 0.1−2.3 μg·mL−1 where Y and X are peak area and plasma concentrations, respectively. After oral administration of BMAP and ethanol extract of AP to rabbits, the plasma concentration-time profiles of andrographolide are presented in Fig. 5 (the content of dehydroandrographolide was too low to detect). It was found that the concentrations of andrographolide in BMAP were higher than that in ethanol extract almost at each time pointand maintained a high blood concentration for a long time. The concentration of andrographolide in BMAP group (with three obvious absorption peaks at 30 min, 1.5 h, and 3 h) declined more slowly after reaching the peak at 3 h compared

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Fig. 4 Representative HPLC chromatograms of microemulsion (A), reference substance of andrographolide and dehydroandrographolide (B), Ethanol extract (C), and BMAP (D). 1. Andrographolide 2. dehydroandrographolide

Fig. 5 Concentration-time curves of andrographolide in BMAP and the ethanol extract

with the ethanol extract group. The concentration of andrographolide in BMAP group maintained at a high level and reached the first peak at about 30 min, which may relate to the absorption in stomach. The second peak appearing at 1.5 h may be produced by the lymphatic absorption of each phase of the BMAP which reached a higher concentration after being absorbed into blood. The third peak of andrographolide concentration after oral administration appeared at 3 h and then waned at a low rate. Throughout the gastrointestinal tract andrographolide and dehydroandrographolide can be absorbed [43]. The different parts of the gastrointestinal tract have different absorption for these two monomer components. What caused the third peak may be that, along with the gradually metabolism in the stomach, the andrographolide reached in the intestines after easily passing through the hydration layer of the gastrointestinal wall because of the low surface tension of the BM, which can contact the intestinal epithelial cell directly, thereby promoting absorption of the drug. Pharmacokinetic parameters are shown in Table 3. The AUC0-7 value of andrographolide in BMAP was higher than that in ethanol extract, which indicated that the oral bioavailability and efficacy of the former were greater than the latter; the possible reason for this result was higher concentration or more ingredients of the BMAP, which needs more pharmacological studies in the future. Furthermore, the peak time of andrographolide in BMAP was shorter than that in ethanol extract and could maintain a high blood concentration.The elimination rate of andrographolide in BMAP was much smaller, indicating that the andrographolide in BMAP can be absorbed quickly by the parietal cells of gastrointestinal and the metabolic rate of drug can be extended appropriately, and that more than one absorption peak represents more absorption mechanisms coexist, allowing the plasma concentration to be maintained at a high level.

Table 3 Pharmacokinetic parameters of BMAP and ethanol extract of AP AUC0→7 /(µg·mL−1·h‒1)

AUC0→∞ /(µg·mL−1·h‒1)

Cmax /(µg·mL−1)

Tmax (h)

K

BMAP

2.267 ± 0.067*

27.156 ± 0.63**

0.301 ± 0.02

0.5 ± 0.02*

0.009 ± 0.00

Ethanol extract

1.606 ± 0.084

4.308 ± 0.44

0.301 ± 0.05

3 ± 0.21

0.067 ± 0.04

EDA0→∞

EDA0→7

6.303

1.420

*

P < 0.05, **P < 0.01 vs ethanol extract

To improve the solubility and the dissolution rate of poorly soluble drugs is an important way to improve the bioavailability of oral administration [44] . After entering the gastrointestinal tract, one part of the drug was mainly transported via intestinal epithelial cells into the portal vein, then entering into the systemic circulation after being metabolized by liver, and another part can be ingested into the lymphatic capillaries through the knot (PP)

on intestinal mucosa and remitted to the thoracic duct before entering into the circulation through jugular vein [45] . In our experiment, andrographolide in BMAP can be absorbed by lymphatic after oral administration and contacted the epithelial cells of gastrointestinal directly after passing through the hydration layer of the gastrointestinal wall, which made this ingredient be absorbed faster.

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Conclusion In our study, cholesterol was used as oil phase, lecithin as surfactant and bile salts as co-surfactant. By combining with polyoxyethylene castor oil, a microemulsion with a good biocompatibility and low toxicity can be prepared. With the methods of mixed design and the mathematical model, the best ratio of the BM was screened and the BMAP with good biocompatibility was prepared successfully. With an average droplet size of 19.12 nm, the BM has good thermodynamic stability under different conditions. Compared with the ethanol extract of AP, the BMAP showed stronger drug potency with a higher blood concentration and better bioavailability after oral administration. In conclusion, some shortcomings in the current extraction methods of AP could be overcome by using BMAP which extracted comprehensively and thoroughly, with higher bioavailability.

Acknowledgements We thank Dr. Andy QIU for proof reading this manuscript.

References [1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

Jang ZO. Process on the chemical compositions of the Andrographis genus plants [J]. Chin Tradit Patent Med, 2011, 33: 1382-1388. Gao XM. Science of Chinese Materia Medica [M]. 2nd edition Beijing: China Press of Traditional Chinese Medicine, 2007: 109-110. Guan C, Li M, Ren QJ, et al. Andrographlide protects mice against severe enterovirus 71 infection by its anti-inflammation and immunomodulation effects [J]. J Immunological, 2013, 29(9): 737-744. RA Burgos, JL Hancke, JC Bertoglio, et al. Efficacy of an Andrographis paniculata composition for the relief of rheumatoid arthritis symptoms: a prospective randomized placebo-controlled trial [J]. Clin Rheumatol, 2009, 28: 931-946. Aggarwal BB, Prasad S, Reuter S, et al. Identification of novel anti-inflammatory agents from ayurvedic medicine for prevention of chronic diseases [J]. Curr Drug Targets, 2011, 12(11): 1595-1653. SP G, Tee W, DS N, et al. Andrographolide protects against cigarette smoke-induced oxidative lung injury via augmentation of Nrf2 activity [J]. Br J Pharmacol, 2013, 168(7): 1707-1718. Lee KC, Chang HH, Chung YH, et al. Andrographolide acts as an anti-inflammatory agent in LPS-stimulated RAW 264.7 macrophages by inhibiting STAT3-mediated suppression of the NF-kappa B pathway [J]. J Ethnopharmacol, 2011, 135(3): 678-684. CV C, Murali B, Deepak M, et al. In vitro comparative evaluation of non-leaves and leaves extracts of Andrographis paniculata on modulation of inflammatory mediators [J]. Antiinflamm Antiallergy Agents Med Chem, 2012, 11(2): 191-197. Chan SJ, Wong WS Fred, Wong Peter TH, et al. Neuroprotective effects of andrographolide in a rat model of permanent cerebral ischaemia [J]. British J Pharm, 2010, 161(3): 668-679.

[10] Raghava NDS, Prasanna RY, Rahul N, et al. Chronic effects of Andrographis paniculata chloroform extract in spontaneously hypertensive rats [J]. J Pharm Sci Res, 2012, 4(8): 1924-1928. [11] Sandborn WJ, Targan SR, Byers VS, et al. Andrographis paniculata extract (HMPL-004) for active ulcerative colitis [J]. Am J Gastroenterol, 2013, 108(1): 90-98. [12] Saranya P, Geetha A. Selvamathy S.M.K. N. A biochemical study on the gastroprotective effect of andrographolide in rats induced with gastric ulcer [J]. Indian J Pharm Sci, 2011, 73(5): 550-557. [13] Harrison J. Hockera, Kwang-Jin Cho, Chung-Ying K. Chen, et al. Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic ras function [J]. N-S Arch Pharmacol, 2013, 110(25): 10201-10206. [14] Guan SP, Tee W, DSW Ng, et al. Andrographolide protects against cigarette smoke-induced oxidative lung injury via augmentation of Nrf2 activity [J]. Brit J Pharmacol, 2013, 168: 1707-1718. [15] Ajit KT, Shyam SC, Vikas K. Neuropsychopharmacology of a therapeutically used Andrographis paniculata extract: a preclinical study [J]. Orient Pharm Exp Med, 2014, 14: 181-191. [16] Zhang Y, Zhang X, Lai XR, et al. Progress on extract technology and the determination of andrographis [J]. Chin Tradit Patent Med, 1993, 15(6): 6-7. [17] Meng CY, Luo JH, Zhang LD, et al. Extraction of total flavanone from common andrographis herb and its effects on scavenging of hydroxyl radicals [J]. Technol Dev Chem Ind, 2006, 35(8): 7-9. [18] Liu L, Fu MZ, Wang X, et al. Progress on pathogen and immune characteristics of porcine reproductive and respiratory syndrome [J]. Prog in Vet Med, 2011, 32(6): 151-155. [19] Huang Q, Tian YH, Liu YM, et al. Microwave-assisted extraction of total flavones from Andrographis Paniculata [J]. Modern Food Sci Tech, 2011, 11: 1372-1374. [20] Peng MW, Wei TY, Chen XG, et al. Study on extraction process of andrographis extracted by internal pressure boiling method [J]. Lishizhen Med Mater Med Res, 2011, 22(9): 2244-2246. [21] Yang T, Sheng HH, Li Y, et al. Optimization of extraction process of andrographispaniculata by central composite design-response sur-face methodology [J]. J Chin Pharm, 2011, 46(3): 208-213. [22] Gao YY, Wei Q, Fan QS, et al. Advances of high-speed countercurrent chromatography on separation of natural products [J]. Food Sci, 2008, 29(2): 461-465. [23] Kang SH. Study on supercritical extraction of Elsholtzia densa Benth [J]. Chin Tradit Patent Med, 2012, 34(5): 955-956. [24] Yang ZB, Long CM, Guo ZY, et al. Analysis of volatile composition of leaf and fruit in Betula luminifera by MAE-HS-SPME [J]. Chin J Exp Tradit Med Form, 2012, 18(1): 56-59. [25] Shi YB, Li HL, Wang HQ, et al. Simultaneous determination of five anthraquinones in a Chinese traditional preparation by RP-HPLC using an improved extraction procedure [J]. J Integrative Med, 2014, 12(5): 455-462. [26] Zhang PX. Study on active ingredients and harmful residues of Chinese medicine extracted by ultrasonic atomizing extraction [D]. Jilin: Jilin University, 2014. [27] Hoar TP, Schulman JH. Transparent water-in-oil dispersions: the oleopathic hydro-micelle [J]. Nature, 1943, 152(102):

– 690 –

LIU Xiao-Yan, et al. / Chin J Nat Med, 2016, 14(9): 683−691

102-103. [28] Jayne ML, Gareth DR. Microemulsion based media as novel drugdelivery systems [J]. Adv Drug Deliver Rev, 2000, 45: 89-121. [29] Mehta SK, Kaur G, Bhasin KK. Entrapment of multiple anti-Tb drugs in microemulsion system: quantitative analysis, stability, and in vitro release studies [J]. J Pharm Sci, 2010, 99(4): 1896-1911. [30] Gomez del Rio JA, DG Hayes. Protein extraction by Winsor-III microemulsion systems [J]. Biotechnol Prog, 2011, 27(4): 1091-1100. [31] Jolivalt C, Minier M, Renon H. Extraction of cytochromec in sodium dodecyl benzenesulfonate microemulsions [J]. Biotechnol Prog, 1993, 9(5): 456-461. [32] Du H, Feng QJ, Yang XZ, et al. “Whole Chinese angelica” microemulsion: its preparation and in vivo and in vitro evaluations [J]. Drug Dev Ind Pharm, 2014, 40(10): 1330-1339. [33] Li R, Wang QS. Evaluated methods and developmental trend of biocompatibility of biomaterials [J]. J Clin Rehabilitative Tissue Eng Res Clin, 2011, 15(29): 5471-5474. [34] Peng ZC. Encyclopedia of Chemical Technology [M]. Beijing: Chemical Industry Press, 1996. [35] Sugipka H, Moroi Y. Micelle formation of sodium cholate and solubilization into the micelle [J]. Biochim Biophys Acta, 1999, 1934(1): 99-110. [36] Muller K. Structural dimorphism of bile salt/lecithin mixed micelles a possible regulatory mechanism for cholesterol solubility in bile X-ray structure analysis [J]. Biochem, 1981, 20(2): 404-414. [37] Aviram Spernatha, Abraham Aserina, Lior Zisermanb, et al.

[38]

[39]

[40]

[41]

[42]

[43]

[44] [45]

Phosphatidylcholine embedded microemulsions: physical properties andimproved Caco-2 cell permeability [J]. J Control Release, 2007, 119: 279-290. TP Formariza, LA Chiavaccia, VHV Sarmentob, et al. Structural changes of biocompatible neutral microemulsions stabilized by mixed surfactant containing soya phosphatidylcholine and their relationship with doxorubicin release [J]. Colloid Surface B, 2008, 63: 287-295. Zhang QZ, Jiang XG, Jiang WM, et al. Preparation of nimedipine-loaded microemulsion for intranasal delivery and evaluation on the targeting efficiency to the brain [J]. Int J Pharm, 2004, 275: 85-96. Matsuda T, Kuroyanacii M, Sugiyama S, et al. Cell differentiation-inducing diterpenes from Andrographis paniculata Nees [J]. Chem Pharm Bull, 1994, 42(6): 1216-1225. Kapil A, Koul IB, Banerjee SK, et al. Antihepatotoxic effects of major diterpenoid constituents of Andrograph is paniculata [J]. Biochem Pharmacol, 1993, 46(1): 182-185. Tripathi GS, Tripathi YB. Choleretic action of andrographolide obtained from Andrographis paniculata Nees [J]. Phytother Res, 1991, 5(4): 176-178. Su H. Study on gastrointestinal absorption of extract of Andrographis Paniculata (Burm.F.) nees. [D]. Henan: Henan University, 2013. Bai ZH, Fang XL. Process on insoluble drugs formulation for oral administration [J]. J Chin Pharm, 2005, 40(15): 1124-1127. Ye ML. Studies on stability and bioavailability of andrographolide microemulsion [D]. Beijing: Beijing University of Chinese Medicine, 2009.

Cite this article as: LIU Xiao-Yan, NIU Xin, FENG Qian-Jin, YANG Xue-Zhi, WANG Dan-Wei, ZHAO Tong, LI Lei, DU Hong. A new biocompatible microemulsion increases extraction yield and bioavailability of Andrographis paniculata [J]. Chin J Nat Med, 2016, 14(9): 683-691.

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