Preparation,evaluation,and in vitro release of chitosan-alginate tanshinone self-microemulsifying sustained-release microcapsules

Abstract

OBJECTIVE:

In this study we explore the method to prepare tanshinone self-microemulsifying sustained-release microcapsules using tanshinone self-microemulsion as the core material, and chitosan and alginate as capsule materials.

METHODS:

The optimal preparation technology of chitosan-alginate tanshinone self-microemulsifying sustained-release microcapsules was determined by using the orthogonal design experiment and single-factor analysis. The drug loading and entrapment rate were used as evaluation indexes to assess the quality of the drug, and the in vitro release rate was used to evaluate the drug release performance.

RESULTS:

The best technology of chitosan-alginate tanshinone self-microemulsifying sustained-release microcapsules is as follows: the concentration of alginate is 1.5%, the ratio of tanshinone self-microemulsion volume to alginate volume to chitosan mass is 1:1:0.5 (ml: ml: g), and the best concentration of calcium chloride is 2.0%. To prepare the microcapsules using this technology, the drug loading will be 0.046%, the entrapment rate will be 80.23%, and the 24-hour in vitro cumulative release rate will be 97.4%.

CONCLUSION:

The release of the microcapsules conforms to the Higuchi equation and the first-order drug release model and has a good sustained-release performance.

Keywords

Tanshinone chitosan-alginate self-microemulsifying sustained-release microcapsules preparation in vitro release

1. Introduction

Tanshinones are a group of fat-soluble components extracted from the lip plant Salvia miltiorrhiza Bge, which mainly include tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone, and hydroxytanshinone. They are the main physiologically active ingredients in Salvia miltiorrhiza [1]. According to pharmacological studies, tanshinone has the effect of treating cardiovascular diseases, and has anti-tumor, antibacterial, and antioxidant effects [2]. However, tanshinone has poor water solubility; therefore, it is necessary to improve its water solubility and absorption through appropriate pharmaceutical preparation methods [3]. There are already some tanshinone preparations on the market: tanshinone IIA sulfonate sodium salt is clinically used as an intravenous infusion to treat coronary heart disease [4]. In addition, there are also studies on the preparation of tanshinone micropowder preparations and microspheres. However, due to the limitation of its administration route and the low drug loading, the existing laboratory preparations have not reached the standard for clinical use [5, 6].

Self-microemulsifying microcapsules are drug-stored microcapsules that are composed of oil, surfactants or cosurfactants, and a small amount of water. The drug is wrapped in the capsule material and can be used as a carrier for hydrophobic, difficult-to-absorb, or easily hydrolyzed drugs [7]. Self-microemulsifying microcapsules can significantly improve the drug bioavailability by increasing the solubility of the drug, reducing the surface tension, forming a hydration layer that easily passes through the stomach and intestinal walls, and increasing the penetration of intestinal epithelial cells [8].

In view of its poor water solubility, self-emulsifying technology was used to enhance this. The prescription of tanshinone self-emulsion is as follows: medium chain triglycerides (MCT); polyoxyethylene 40 (RH40) hydrogenated castor oil; and 1, 2-propanediol (1, 2-PG) $=$ 2:2:1 (w/w/w). Since most of the self-emulsifying agents are oil-like, solid preparations can improve the clinical compliance; therefore, they are prepared into microcapsules.

2. Methods

2.1 Materials

The materials used in this test included the following: tanshinone IIA reference substance (China Institute for the Control of Pharmaceutical and Biological Products); sodium alginate (Qingdao Jingyan Biotechnology Development Co., Ltd., China); tanshinone extract (Chongqing Institute of Daily Chemical Industry, containing 20.14% tanshinone IIA); MCT, RH 40 (Gattefosse, France); 1, 2-PG (Chengdu Kelong Chemical Reagent Factory, China); methanol gas chromatographic pure, and the rest were analytically pure.

2.2 Instruments

The instruments used in this test included the following: high performance liquid chromatograph (HPLC, Agilent 1200 series, VWD detector 190–600 nm, online degassing, binary pump, Agilent, France); electronic balances (1/10,000, 1/100,000, Sartorius, Germany); SK-1 fast mixer (Jintan Chengdong Xinrui Instrument Factory, China); HZ-881S desktop water bath thermostatic oscillator (Taicang Experimental Equipment Factory, Jiangsu Province, China); DF-101 S collector constant temperature heating magnetic stirrer (Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd., China); and RC 806 dissolution tester (Tianjin Tianda Tianfa Technology Co., Ltd., China).

2.3 Determination method of tanshinone IIA

2.3.1 Chromatographic conditions

The chromatographic conditions included the following: chromatographic column: Symmetrix ODS-AQ (4.6 mm $\times$ 250 mm, 5 $\mu$ m); mobile phase: methanol-water (75:25); flow rate: 1.0 ml $\cdot$ min ${}^{-1}$ ; detection wavelength: 270 nm; column temperature: 30 ${}^{\circ}$ C; injection volume: 10 $\mu$ l.

2.3.2 Solution preparation

The preparation of the reference solution: the appropriate amount of tanshinone IIA reference substance was accurately weighed and combined with an appropriate amount of methanol. A reference solution that 1 ml contains 1.01 mg of tanshinone IIA was then prepared.

The preparation of the negative test solution: an appropriate amount of blank self-microemulsifying microcapsules were crushed, and 0.063 g were weighed and diluted with ethyl acetate to make 25 ml.

The preparation of the test solution: an appropriate amount of tanshinone IIA self-emulsifying microcapsules were crushed, and 0.063 g were weighed and diluted with ethanol to make 25 ml.

The preparation of the tanshinone extract solution: an appropriate amount of tanshinone extract was weighed and dissolved with an accurate amount of methanol.

3. Results

3.1 Specificity investigation

A negative test solution, reference solution, test solution, and tanshinone extract solution were taken respectively and injected into the liquid chromatograph, according to the above chromatographic conditions – the results are shown in Fig. 1. The results revealed that the excipients did not interfere with the determination of tanshinone IIA.

Figure 1.

HPLC.

3.2 Preparation of standard curve

The tanshinone IIA reference solution was prepared into a series of mass concentration reference solutions, injected into HPLC, measured, and the peak area was recorded. Linear regression analysis was performed: the abscissa (x) is the mass concentration of the reference substance (mg/ml) and the ordinate (y) is the peak area. The standard curve equation was as follows: y $=$ 36, 321.6, x $=$ 10, 328, r $=$ 0.9999. The result revealed that, in the range of 2.02–101.00 mg/ml, there was a good linear relationship between the concentration of tanshinone IIA and its peak area.

3.3 Precision test

The reference solution was taken, injected continuously six times, and calculated. This resulted in the relative standard deviation (RSD) being 0.18%, suggesting that the precision of the instrument was good.

3.4 Stability test of test solution

The same test solution was taken, placed in darkness for preservation, and injected at 0, 2, 4, 8, 12, and 24 hours, respectively. Result: within 24 hours, the RSD of tanshinone IIA in the test solution was 0.57%. These results indicated that tanshinone IIA was stable within 24 hours.

3.5 Repeatability test

An appropriate amount of tanshinone IIA self-microemulsifying microcapsules were crushed, prepared into six copies of the test solution (according to the preparation method of the test solution), and measured. Result: the RSD of tanshinone IIA in the six copies of the test solution was 0.46%. These results indicated that the repeatability was good.

3.6 Recovery rate test

An appropriate amount of tanshinone IIA self-microemulsifying microcapsules were crushed, and 0.35 g were accurately weighed and combined with 0.1 ml of tanshinone IIA reference solution. This was dispersed with an appropriate amount of ethanol, diluted to 25 ml, divided into six copies equally, and measured. Result: the average recovery rate was 100.08%. These results indicated that the accuracy of the method was good.

3.7 Determination of drug loading and entrapment rate of tanshinone IIA self-microemulsion microcapsules

Drug loading $=$ weight of drugs in microcapsule/total weight of microcapsule $\times$ 100%.

Entrapment rate $=$ weight of drugs in microcapsule/all drugs $\times$ 100%.

3.8 Preparation of tanshinone IIA self-microemulsifying microcapsules

Investigation of the dosage of chitosan: it was revealed in the pre-experimental study that, when mixed with alginate, tanshinone IIA self-microemulsion is still oil-like and cannot be completely dried; therefore, chitosan is added for adsorption. An appropriate volume of tanshinone IIA self-microemulsion was mixed with varying proportions of chitosan. Result: when the ratio of tanshinone IIA self-microemulsion to chitosan was 1:0.4 (ml: g), chitosan could completely adsorb tanshinone IIA self-microemulsion.

Test operation of preparation of tanshinone IIA self-microemulsifying microcapsules: alginate and chitosan were mixed in proportion to prepare a solution. This was then mixed evenly with tanshinone IIA self-microemulsion, dropped into calcium chloride solution using a 9 needle, and slowly stirred with a magnetic stirrer at the speed of approximately 30 drops/min. The drop distance (the height between the needle tip and the liquid surface of calcium chloride solution) was 10 cm. It was then solidified for ten minutes and filtrated, following which dark red microcapsules were obtained. These were dried at 45 ${}^{\circ}$ C and the product was obtained. The results are shown in Fig. 2.

3.9 Optimization of tanshinone IIA self-microemulsifying microcapsules by orthoplan

An L9 (3 ${}^{4}$ ) orthogonal experiment was conducted considering the following factors: the concentration of alginate; the volume ratio of alginate to indirubin; and the concentration of calcium chloride. Drug loading and the entrapment rate were the main investigation indexes to optimize the preparation process of tanshinone IIA self-microemulsifying microcapsules. The factor levels are shown in Table 1.

Table 1
Factor level table

Level	Factors
	A the concentration of alginate (%)	B the volume ratio of alginate to indirubin (ml:ml)	C the concentration of calcium chloride (%)	D blank
1	0.5	1:1	1
2	1.5	1:2	2
3	3	1:3	3

Figure 2.

Tanshinone IIA self-microemulsifying microcapsules.

The results revealed that the influencing factors A and B had significant effects on the process of tanshinone IIA self-microemulsifying microcapsules; therefore, the best technological conditions are as follows: A2B1C2 – the concentration of alginate is 1.5%; tanshinone IIA self-microemulsion is mixed with alginate solution in a volume ratio of 1:1; and the concentration of calcium chloride solution is 2.0%. Detailed results are shown in Tables 2 and 3.

Table 2

Results of orthogonal experiment

No.	A	B	C	D	Drug loading content (%)	Encapsulation efficiency (%)	The comprehensive score
1	1	1	1	1	0.051	40.27	75.42
2	1	2	2	2	0.038	38.22	61.38
3	1	3	3	3	0.017	23.37	31.42
4	2	1	2	3	0.046	79.21	95.10
5	2	2	3	1	0.022	67.32	64.06
6	2	3	1	2	0.025	40.05	49.79
7	3	1	3	2	0.019	64.96	59.63
8	3	2	1	3	0.021	48.06	50.93
9	3	3	2	1	0.014	43.51	41.19
K1	56.073	76.717	58.713	60.223
K2	69.650	58.790	65.890	56.933
K3	50.583	40.800	51.703	59.150
Range	19.067	35.917	14.187	3.290

Note: The comprehensive score: The weight coefficients of drug loading and entrapment efficiency were 0.5; The comprehensive score: Drug loading/maximum drug loading * 100 * 0.5 $+$ entrapment efficiency/maximum entrapment efficiency * 100 * 0.5.

Table 3

Analysis of variance table

Factors	Sum of squares of deviations	Degrees of freedom	F value	F critical value	Significance
A	578.004	2	34.222	19.000	*
B	1935.012	2	114.566	19.000	*
C	301.906	2	17.875	19.000
D	16.890	2	1.000	19.000
Errors	16.89	2

3.10 Validation of the preparation process of tanshinone IIA self-microemulsifying microcapsules

Tanshinone IIA self-microemulsifying microcapsules were prepared repeatedly three times; the results are shown in Table 4.

Table 4
Process verification

No.	Drug loading content(%)	Encapsulation efficiency(%)
1	0.046	81.05
2	0.047	79.26
3	0.046	80.38
The average	0.046	80.23

In summary, the preparation process of tanshinone IIA self-microemulsifying microcapsules was determined as follows: tanshinone IIA self-microemulsion-chitosan-1.5% sodium alginate $=$ 1:0.5:1 (ml: g: ml), were shaken to mix, then dropped with a 9 needle into calcium chloride solution and slowly stirred with a magnetic stirrer at the speed of approximately 30 drops/min. The drop distance (the height between the needle tip and the liquid surface of calcium chloride solution) was 10 cm. It was then solidified for ten minutes and filtrated, following which dark red microcapsules were obtained. These were dried at 45 ${}^{\circ}$ C, and the product was obtained. The size of tanshinone IIA self-microemulsifying microcapsules was approximately 0.3 cm.

3.11 Preliminary study on in vitro drug release of tanshinone IIA self-microemulsifying microcapsules

According to the Chinese Pharmacopoeia 2015 edition, Volume IV, General Rule 0931, Approach II, 900 ml of water, treated by degassing, was used as the release mediator. The temperature was 37 ${}^{\circ}$ C and the rotational speed was 100 r/min. The microcapsules prepared were placed in a large cup and samples were taken at 0, 0.5, 1, 2, 3, 4, 6, 12, and 24 hours, respectively. The sample size was 5 ml and was mixed with the same volume of mediator and at the same temperature simultaneously. The filtrates were then continuously taken and measured. The cumulative drug release curve was drawn and fitted – the results are shown in Table 5.

Table 5
The results of the cumulative drug release curve ( $n=$ 3)

	Equation	$R^{2}$
Zero order kinetics	Q $=$ 5.9547t	0.3739
One order kinetics	ln (1-Q) $=-$ 0.1831t	0.8659
Higuchi equation	Q $=$ 24.549t ${}^{1/2}$	0.8768

Note: Q is the cumulative release degree (%); T is time (h).

The results reveal that the release curve of the microcapsules in water was well fitted by the first-order equation and Higuchi equation. These results indicate that the microcapsule has a certain sustained-release effect.

4. Discussion

Although tanshinone has anti-cardiovascular disease, anti-tumor, antibacterial, and antioxidant effects, the water solubility of most of its active ingredients is extremely poor, and the in vivo absorption is not good [9, 10, 11]. At present, due to the limitation of administration routes and low drug loading, the existing tanshinone preparations in terms of in vivo absorption have not reached the ideal state [12]. Self-microemulsifying microcapsules can be used as carriers of hydrophobic, difficult-to-absorb, or easy-to-hydrolyze drugs. These form a hydration layer and can easily pass through the walls of the stomach and intestine by improving the solubility of the drug and reducing the surface tension. This can increase the penetration of intestinal epithelial cells to significantly improve the drug bioavailability [13, 14, 15, 16]. Therefore, tanshinone self-microemulsifying microcapsules were prepared by the self-microemulsion method.

After the measurement method for the content of tanshinone IIA was established, the preparation of tanshinone IIA self-microemulsifying microcapsules were investigated. It was revealed in the pre-experimental study that, because tanshinone IIA self-microemulsion was oil-like, it could not be completely dried after mixing with alginate; therefore, it is necessary to add chitosan for adsorption [17]. Once the ratio of tanshinone IIA self-microemulsion to chitosan was 1:0.4 (ml: g), chitosan could completely adsorb tanshinone IIA self-microemulsion. Therefore, this ratio was used in subsequent experiments. In this study, the preparation process was further optimized by orthoplan to obtain self-microemulsifying microcapsules with high drug loading and stability. The results reveal that the concentration of alginate should be 1.5%, tanshinone IIA self-microemulsion should be mixed with alginate solution in a volume ratio of 1:1, and the concentration of calcium chloride solution should be 2.0%.

During the preparation process of tanshinone IIA self-microemulsifying microcapsules, the emulsifiable oil method was used [18]. The mixed oil was dropped with a 9 needle into calcium chloride solution and slowly stirred with a magnetic stirrer at the speed of about 30 drops/min. It was then solidified for ten minutes and filtrated, following which self-microemulsifying microcapsules were obtained. This method can obtain self-microemulsifying microcapsules with uniform particles and a high entrapment rate [19]. The present study also revealed that the entrapment rate of microcapsules prepared was 80.23%. However, the average drug loading was 0.046%, which was relatively low. The reason for this may be that the solubility of tanshinone IIA in water is low. It is prepared into microcapsules and then prepared into self-microemulsion. The drug loading of self-microemulsion is low [20]; therefore, increasing this is key to improving the drug loading of microcapsules.

The investigators also conducted a preliminary study on the in vitro drug release of microcapsules. The results revealed that the release curve of the microcapsules in water was well fitted by the first-order equation and Higuchi equation. These results indicate that the microcapsule has a certain sustained-release effect. In this study, chitosan was used to prepare the microcapsules. The drug could be adhered to the surface of the microcapsules and was not easy to diffuse into the solution directly or rapidly. In future, the sustained-release and adhesion of chitosan in vivo also needs to be further explored [17].

To avoid limiting this study, the drug loading needs to be improved. Additionally, it is necessary to study the absorption and drug distribution in vivo in subsequent animal experiments, thus enabling the preparation of tanshinone self-microemulsifying sustained-release microcapsules for use in clinical practice.

5. Conclusion

The best technology of chitosan-alginate tanshinone self-microemulsion sustained-release microcapsules is as follows: the concentration of alginate is 1.5%, the ratio of tanshinone self-microemulsion volume to alginate volume to chitosan mass is 1:1:0.5 (ml: ml: g), and the best concentration of calcium chloride is 2.0%. For the microcapsules prepared using this technology, the drug loading is 0.046%, the entrapment rate is 80.23%, and the 24-hour in vitro cumulative release rate is 97.4%. The release of the microcapsules conforms to the Higuchi equation and the first-order drug release model, and such microcapsules have a good sustained-release performance.

Footnotes

Acknowledgments

The authors would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

Conflict of interest

The authors declare that they have no competing interests.

Funding

None to report.

References

Long

Yang

Xiang

Zhang

Wan

Liu

Peng

. Nose to brain drug delivery – a promising strategy for active components from herbal medicine for treating cerebral ischemia reperfusion. Pharmacol Res. 2020; 159: 104795.

Wang

Yang

Liu

Gao

. Pharmacological properties of tanshinones, the natural products from salvia miltiorrhiza. Adv Pharmacol. 2020; 87: 43–70.

Zhou

Zhao

Zhang

Chen

Tang

. Sodium tanshinone IIA sulfonate: a review of pharmacological activity and pharmacokinetics. Biomed Pharmacother. 2019; 118: 109362.

Jiang

Gao

Huang

. Tanshinones, critical pharmacological components in salvia miltiorrhiza. Front Pharmacol. 2019; 10: 202.

Peng

Jiang

Farhan

Lazarovici

Chen

Zheng

. Anti-inflammatory effects of traditional Chinese medicines on preclinical in vivo models of brain ischemia-reperfusion-injury: prospects for neuroprotective drug discovery and therapy. Front Pharmacol. 2019; 10: 204.

Liu

Wang

Duan

Zhang

Liu

Zhao

. Efficacy of danshen class injection in the treatment of acute cerebral infarction: a bayesian network meta-analysis of randomized controlled trials. Evid Based Complement Alternat Med. 2019; 2019: 5814749.

Midha

Nagpal

Singh

Aggarwal

. Prospectives of solid self-microemulsifying systems in novel drug delivery. Curr Drug Deliv. 2017; 14(8): 1078–1096.

Gibaud

Attivi

. Microemulsions for oral administration and their therapeutic applications. Expert Opin Drug Deliv. 2012; 9(8): 937–51.

Qiu

Guo

Wang

Zhang

Wang

Chen

. Potential pharmacokinetic herb-drug interactions: have we overlooked the importance of human carboxylesterases 1 and 2? Curr Drug Metab. 2019; 20(2): 130–137.

10.

Wei

Zhang

Lei

Yang

Wang

Han

Xia

Yang

. Ethnopharmacology, phytochemistry, and pharmacology of Chinese salvia species: a review. J Ethnopharmacol. 2018; 225: 18–30.

11.

Sahoo

Dandapat

Dash

Kanhar

. Features and outcomes of drugs for combination therapy as multi-targets strategy to combat alzheimer’s disease. J Ethnopharmacol. 2018; 215: 42–73.

12.

Parvez

. Natural or plant products for the treatment of neurological disorders: current knowledge. Curr Drug Metab. 2018; 19(5): 424–428.

13.

Huang

Zhang

Shen

Gao

. Controlled release of the nimodipine-loaded self-microemulsion osmotic pump capsules: development and characterization. AAPS Pharm Sci Tech. 2018; 19(3): 1308–1319.

14.

Ćetković

Cvijić

Vasiljević

. In vitro/in silico approach in the development of simvastatin-loaded self-microemulsifying drug delivery systems. Drug Dev Ind Pharm. 2018; 44(5): 849–860.

15.

Zhao

Yuan

Wang

Bao

. Preparation and evaluation of valsartan by a novel semi-solid self-microemulsifying delivery system using gelucire 44/14. Drug Dev Ind Pharm. 2016; 42(10): 1545–52.

16.

Xiumin

Man

Minzi

Yinghua

Dongqin

. The in vitro and in vivo evaluation of fenofibrate with a self- microemulsifying formulation. Curr Drug Deliv. 2015; 12(3): 308–13.

17.

Peers

Montembault

Ladavière

. Chitosan hydrogels for sustained drug delivery. J Control Release. 2020: S0168-3659(20)30346-1.

18.

Zvonar

Bolko

Gašperlin

. Microencapsulation of self-microemulsifying systems: optimization of shell-formation phase and hardening process. Int J Pharm. 2012; 437(1–2): 294–302.

19.

Gibaud

Attivi

. Microemulsions for oral administration and their therapeutic applications. Expert Opin Drug Deliv. 2012; 9(8): 937–51.

20.

Wang

Gan

Zhang

. Novel gastroretentive sustained-release tablet of tacrolimus based on self-microemulsifying mixture: in vitro evaluation and in vivo bioavailability test. Acta Pharmacol Sin. 2011; 32(10): 1294–302.

Preparation,evaluation,and in vitro release of chitosan-alginate tanshinone self-microemulsifying sustained-release microcapsules

Abstract

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Methods

2.1 Materials

2.2 Instruments

2.3 Determination method of tanshinone IIA

2.3.1 Chromatographic conditions

2.3.2 Solution preparation

3. Results

3.1 Specificity investigation

3.3 Precision test

3.4 Stability test of test solution

3.5 Repeatability test

3.6 Recovery rate test

3.7 Determination of drug loading and entrapment rate of tanshinone IIA self-microemulsion microcapsules

3.8 Preparation of tanshinone IIA self-microemulsifying microcapsules

3.9 Optimization of tanshinone IIA self-microemulsifying microcapsules by orthoplan

Table 1 Factor level table

Table 4 Process verification

Table 5 The results of the cumulative drug release curve ( n = 3)

5. Conclusion

Footnotes

Acknowledgments

Conflict of interest

Funding

References

Table 1
Factor level table

Table 4
Process verification

Table 5
The results of the cumulative drug release curve ( $n=$ 3)