The Development of Centellaasiatica Extract-Loaded BSA Nanoparticles Production to Improve Bioavailability

Introduction

C. asiatica(Bao-bog) is herbal in Asia. In Ayurvedic, an Indian system of medicine, C. asiaticais used for the treatment of leprosy, insanity, asthma, ulcers, eczema, skin and gastrointestinal disorder, arthritis, varicose vein and high blood pressure¹. The major biologically active compounds of C. asiaticaextract are monoterpenes, sesquiterpene, and triterpenoids². It was found that the chemical complexity of the herbal extracts seem to be important for the bioavailability. Even though, C. asiaticanano powder gave higher yield of active compound (Asiatic acid) than natural C. asiatica powder³there have been some limitations in C. asiatica crude extracts that it showed their extra ordinary potential in-vitro but less or no in-vivo activity due to their poor lipid solubility and improper molecular size, resulting in poor absorption, slow delivery, poor dosing and poor bioavailability. Especially, it is also difficult for water-soluble biological active compounds to enter through cell membrane of both human and pathogenic microorganism, which has hydrophobic characteristics. Albumin is a protein that can be obtained from a variety of sources, including egg white (ovalbumin), bovine serum albumin (BSA), and human serum albumin (HSA). Albumin is the major soluble protein of the circulating system and involved in the maintenance of osmotic pressure and binding and transport of nutrients to the cells. Albumin can soluble in water and diluted salt solution very well⁴. The high solubility of albumin (up to 40% w/v). Albumin is stable in the pH range of 4 to 9 and can be heated at 60°C up to 10 hours without any deleterious effects and at pH 7.4 makes it an attractive macromolecular carrier capable of accommodating a wide variety of molecule⁵. The development of CBNP are the guild line to overcome these problems. Not only improve drug delivery system but the CBNP also reduce cost from purification steps, and can be applied to use in high valued industry products. Therefore, the objective of this research is to develop CBNP on antibacterial activity, antioxidant activity, and nanoparticles properties.

Materials Methods

Preparation of Sample

C. asiaticawas purchased from local markets in Bangkok, Thailand. The aerial part of C. asiaticawas used. Fresh C. asiatica were washed with tap water and cut into small pieces. Then it was air dried in oven (Memmert UM500) at 45°C. The dried samples was finely ground into powder. The powder were kept at 4°C before used.²

Preparation of C. Asiatica Crude Chloroform Extract

C. asiaticawas extracted with chloroform using 1:10 ratio (g/ml). The mixture was macerated at room temperature, 120 rpm, for 48 hours and then it was filtered using whatman filter paper no.4. The crude extract was concentrated using rotary evaporators at 45°C (BUCHI Rotavapor R-205) are then it was kept at -20°C before use.² The C. asiaticacrude chloroform extracts was further used for preparation of CBNP.

Preparation of C. Asiatica Extract-Loaded BSA Nanoparticles

CBNP was prepared by the desolvation method⁶. The 100 mg of BSA were dissolved in 1 ml of sodium chloride solution (10mM). Then, 8.0 ml of ethanol was added dropwise into the BSA solution under magnetic stirring (400 rpm) at room temperature. Subsequently, the as-prepared BSA nanoparticles were cross-linked with 0.2% glutaraldehyde (GA). Then, C. asiaticacrude chloroform extract was added into the solution for 24 hours at different ratio of C. asiatica to BSA (1:2, 1:3, and 1:4) in the preparation of CBNP. The particles were centrifuged and washed with distilled water. The centrifuged particles were resuspended and disperse in 2% mannitol, then freeze-dried for 24 hours. The dried nanopowder werekept at room temperature before use.

Antimicrobial Activity

The modified agar well diffusion method² is used. The 100 μl of bacteria (approximately 1.5 × 10⁸ CFU/ml) is swab on Mueller-Hinton agar (MHA) plate. The 50μl ofC. asiaticacrude chloroform extract and CBNP at concentration 100, 200, and 300 µg/ml diluted with distilled water were used to test antibacterial activity against Escherichia coli ATCC25822, Salmonella entericaTyphimurium U302 (DT104b), S. entericaEnteritidis (human), S. enterica 4,5,12:i:-  (human) US clone, and  Bacillus cereus. The 20μl of 50 mg/ml penicillin G was used as positive control. The inhibition zones were measured to determine the effectiveness of the C. asiaticacrude extract and CBNP against each bacterium. The experiment was done in duplicate and three replication independently.

Antioxidant activity by Total Phenolic Content

The modified Folin–Ciocalteu method⁷was used for total phenolic content determination of C. asiaticacrude chloroform extract and CBNP. The 20 μl of 10 mg/ml C. asiaticacrude chloroform extract and CBNP was added to 1.58 ml distilled water and 100 μlFolin–Ciocalteu phenol reagent. The mixture was then allow to stand for 8 minutes 30 seconds and 300 μl saturated sodium carbonate solution was added to the mixture. Then the mixture was incubated without light at room temperature for 30 minutes and observed optical density (OD) at 765 nm. The result were expressed as microgram garlic acid equivalent (µgGAE/ml). The experiment was done in triplicate and three replicationindependently.

Antioxidant Activity by Ferric Reducing Antioxidant Potential Assay (FRAP)

The modified ferric reducing antioxidantpotential assay⁸ was used to determine FRAPvalue of C. asiatica crudechloroform extract and CBNP. The FRAPreagent was prepared using 300 mmol sodiumacetate buffer at pH 3.6, 20 mmol iron chloride and10 mmol 2,4,6-tripyridyl-s-triazine dissolved in 40mmol hydrochloric acid at a ratio of 10:1:1 (v:v:v).The reagent was incubated at 37°C for 10 minutes before use. The 20μl of 1 mg/ml the extract and CBNP wasadded, followed by adding 1000μl of FRAP reagentvigorously and kept in the dark for 30 minutes. The optical density (OD)of this mixture was measured at 593nm. FRAP values were expressed as mmol Fe²⁺/mg of sample. All measurements were done intriplicate and three replications independently.

Antioxidant Activity by DPPH Radical Scavenging Activity

The modified DPPH radical scavenging activity⁹was used for percentage DPPH radical scavenging determination.The 100 μl of 1 mg/ml C. asiatica crude chloroform extract and CBNP were mixed with 3.9 ml DPPH reagent (50 μM). The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 30 minutes. The optical density (OD) was measured at 517 nm. The results were expressed as percentage reduction of DPPH¹⁰.

Where A₀ is the initial absorbance and A_c is the value for added sample concentration c.All measurements were done intriplicate and three replications independently.

Entrapment Efficiency and loading Efficiency

C. asiatica crude chloroform extract was run absorbance spectrum to find the best the wavelength (λmax) at which the absorbance is the greatestby UV-vis spectrophotometer. The 2 mg CBNP were dissolved in 1 ml methanol and gently shaken for 24 hours at 37 °C to completely extract C. asiatica crudechloroform extract to methanol¹¹.Then the solutions were centrifuged at 12000 rpm for 10 min, and the supernatant was kept and measured optical density (OD) by a UV-vis spectrophotometer at λmax. The amount of C. asiatica crudechloroform extract entrapped and loaded in CBNP is express as entrapment efficiency and loading efficiency calculated as follows¹¹:

All measurements were done intriplicate and three replications independently.

Solubility and Stability

To compare the solubility of C. asiatica crude chloroform extract before and following the encapsulation process, saturation solubility is determined¹¹. Excessive samples (C. asiatica crudechloroform extract and CBNP) were dispersed into 1 mL water and at 200 rpm, 37 °C. After 24 hours, samples were taken out and filtered through a 0.22 μm Millipore membrane. Filtrate was diluted appropriately, and the optical density (OD) were measured by a UV-vis spectrophotometer at λmax.

The 1 mg/mL of CBNP in phosphate buffer solution (0.01M, pH7.4) were incubated at 200 rpm, 37 °C, for 24 hours. At designated time points (0, 0.5, 1, 2, 3, 4, 5, 6 hours), the mixture was sampled and the optical density (OD) was measured by UV-visspectrophotometer at λmax¹¹. The stability of CBNP is calculated as follows:

Where C₀ is the initial absorbance and C_t is theabsorbance of the sample at time point.All measurements were done intriplicate and three replications independently.

Release kinetic in Vitro

Release kinetic¹¹ methodology was modified. The release of C. asiatica crude chloroform extract from CBNP was done by dissolving 20 mg of CBNP in 15 ml artificial gastric juice (0.01 M PBS pH 2.0) and intestinal juice without enzymes (0.01 M PBS pH 7.4). The mixture is incubated at 37 °C at 200 rpm. At designated time points (0, 0.5, 1, 2, 3, 4, 5, 6 hours),mixture is sampled and centrifuged at 3000 rpm for 10 min. The pellet is resuspended in 100 μL of methanol to determine the amount of C. asiatica crude chloroform extract released by measuring optical density (OD) by UV-visspectrophotometer at λmax..All measurements were done intriplicate and three replications independently.

Statistical Analysis and Experimental Design

All experiments were conducted in three replications and statistical analysis wasaccomplished using ANOVA with Duncan’s multiple range tests (p < 0.05) by SAS software version 9.3.

Results and Discussion

Antimicrobial Activity

CBNP were prepare by a desolvationmethod⁶. Nanoparticles are taken up by cells more efficiently than larger micromolecules and therefore, could be used as effective transport and delivery systems. The strategy of applying nanotechnology to plant extracts has been widely used, because nanostructure systems could increase effect of action of plant extracts, promote sustained release of active constituents, reduce the required dose, decrease side effects, and improve activity^{12, 13}.There are three different of the nanoparticles preparative. The different between these three nanoparticles were ratio of C. asiaticacrude chloroform extracts to BSA 1:2, 1:3, and 1:4. The well agar diffusion method² was used for evaluating antibacterial activity of CBNP and C. asiatica crude chloroform extracts with different concentration (100, 200, and 300 µg/ml) against five food borne pathogens (Escherichia coli ATCC25822, Salmonella entericaTyphimurium U302 (DT104b), S. entericaEnteritidis (human), S. enterica 4,5,12:i:-  (human) US clone, and  Bacillus cereus).

Table 1: Antibacterial activity as inhibition zone (cm) of CBNP and crude extract against different microorganisms Click here to View table

 

Note: Different capital letter (A, B, C) superscript within a row showed significant different at p<0.05.Different small letter (a, b, c) superscript within a column showed significant different at p<0.05.

The results of CBNP and C. asiaticacrude chloroform extracts antibacterial activity were interpreted by using  Randomized Complete Block Design (RCBD) with Duncan’s multiple range tests in SAS program version9.3 as showed in table1. It was found that antibacterial activities trend of CBNP increased from C. asiaticacrude chloroform extracts about 2-3 times significantly(p<0.05). C. asiatica crude chloroform extracts have low antibacterial activity because there is a hydrophilic active compound so, it is difficult for water-soluble biological active compounds to permeate through cell membrane, which has hydrophobic characteristics. But,  bovine serum albumin (BSA) of CBNP is the protein can attract macromolecular and carry variety of molecule of active compound⁵.Albumin nanoparticles are formed by linkage of amino acid by electrostatic and attraction and covalent linkage reagent (e.g. glutaraldehyde, genipin) and can be prepare to a size of between 100-200 nm¹⁴. It also can readily bind and release small molecule⁵. All of these reasons made it can increase an efficiency of absorption to the cells. Moreover, CBNP tend to have more significantly effect on gram-negative bacteria (E. coli ATCC25822, S.entericaTyphimurium U302 (DT104b), S. entericaEnteritidis (human), and S. enterica 4, 5, 12: i: – (human) US clone)than gram-positive bacteria (B. cereus). Gram-positive and gram-negative bacteria have difference structural in cell wall composition¹⁵. In Gram positive bacteria, there is an inner membrane which is surrounded by a thick cell wall made of peptidoglycan¹⁶. In Gram negative bacteria, there is an inner membrane followed by a thin peptidoglycan layer and an outer membrane¹⁶. Furthermore, it was found that the different ratio of C. asiatica crude chloroform extracts: BSA and concentration showed no significant difference in antibacterial activity of CBNP against all pathogens except S. entericaEnteritidis (human) and B. cereus (P < 0.05).The highest antibacterial active was 1.07±0.46 cm using CBNP ration 1:4, 200 µg/ml against S. entericaEnteritidis (human). The encapsulation process of CBNP can improve antibacterial activity of C. asiatica crude chloroform extractsagainst the pathogens.

Antioxidant Activity

The antioxidant activity can be measured by the ability of the compound that can catch free radicals compounds by scavenging or trapping methods¹⁷. Herb and spice are rich in phenolic compounds¹⁸ which are an active compound that have antioxidant properties as protective agent against free radical compound¹⁹.The majorbiologically active compounds of C. asiatica extract are monoterpenes, sesquiterpene,triterpenoids², polyphenols²⁰ and triterpenes^21. In addition toterpenoids, it also contains high total phenolic contents which contributed by the flavonoidssuch as quercetin, kaempherol, catechin, rutin, apigenin and naringin and volatile oils such ascaryophyllene, farnesol and elemene^{20, 22}.The antioxidant activity of C.asiatica may be due to the reduction of hydroperoxides, inactivation of free radicals, chelation of metal ions or combinations²⁰. The antioxidants present in theC. asiatica may have different functional properties, such as reactive oxygen speciesscavenging (quercetin and catechins) ²³, inhibition of the generation of free radicals and chain-breaking activity, e.g. p-coumaric acids²⁴ and metalchelation²⁵.

Table 2: Total phenolic content , Ferric reducing antioxidant potential, and DPPH radical scavenging of CBNP and C. asiaticacrude chloroform extract

CBNP with different ratio between Crude:BSA and crude	Total phenolic content  (µg GAE/mg dried weight)	FRAP (mmol Fe2+/mg dried weight)	DPPH (% reduction of DPPH)
1:2	14.59 ± 6.74A	0.53 ± 0.16B	11.55 ± 4.70B
1:3	13.15 ± 5.94A	0.49 ± 0.06B	13.57 ± 8.99B
1:4	13.15 ± 7.13A	0.50 ± 0.01B	5.76 ± 10.44B
crude	13.15 ± 1.62A	1.00 ± 0.15A	29.44 ± 8.20A

Note: Different superscript within a column showed significant different at p<0.05

 

The modified Folin–Ciocalteu method⁷, Ferric reducing antioxidant potential assay⁸ and DPPH radical scavenging⁹ were used for evaluating antioxidant activity of CBNP and C. asiatica crude chloroform extracts. The results were interpreted by using Randomized Complete Block Design (RCBD) withDuncan’s multiple range tests in SAS program version9.3as showed in table2.The phenolic compound was bind to BSA mainly by hydrogen bond, electrostatic, and hydrophobic interaction. Molecular structure and the number of hydroxyl group of phenolic compound are also the factor that effect phenolic compound binding with BSA²⁶. Total phenolic content of CBNP and crude extract were determined in comparison with standard garlic acid and the results were expressed in terms of µg GAE/mg dried weight. The highest antioxidant activity represented by the amount of phenolic content of CBNP was 14.59±6.74 µgGAE/mg using ratio 1:2.The amount of phenolic content of CBNP did not show significant different between ratio 1:2 and others (P < 0.05).  For Ferric reducing antioxidant potential assay and DPPH assay, crude chloroform extract showed significantly higher activity than CBNP were 1.00 ± 0.15 mmol Fe²⁺/mg and 29.44 ± 8.20 % DPPH radical scavenging respectively (P < 0.05).After process of nanoparticles, C.asiatica loss of its activity in ferric reducing antioxidant potential assay and DPPH radical scavenging. Because of the active compound maybe are already bind with protein and changed the structural to be inactive form.

Table 3: Entrapment efficientcy, loading efficiency, and solubility of CBNP and C. asiaticacrude chloroform extract

CBNP with different ratio between Crude:BSA and crude	Entrapment efficiency (%)	Loading Efficiency (%)	Solubility (µg/ml)
1:2	42.77 ± 4.23B	41.28± 8.91A	203.58 ± 113.81A
1:3	59.48 ± 8.90B	36.60 ± 7.56A	172.59 ± 13.53A
1:4	96.94 ± 1.48A	42.05 ± 5.68A	168.86 ± 78.78A
crude			244.34 ± 17.10A

Note: Different superscript within a column showed significant different at p<0.05

Entrapment Efficiency and loading Efficiency 

The entrapment efficiency and loading efficiency were calculated using equations 2 and 3 mentioned above. The results were interpreted by using Randomized Complete Block Design (RCBD) withDuncan’s multiple range tests in SAS program version9.3as showed in table3. CBNP using ratio 1:4 has entrapment efficiency significantly higher than ratio 1:3 and 1:2 which were 96.94 ± 1.48%, 59.48 ± 8.90%, and 42.77 ± 4.23% respectively (p<0.05). There are no significantly difference in entrapment efficiency between CBNP using ratio 1:3 and 1:2 (p<0.05). For loading efficiency, there are no significantly different among ratio of CBNP (p<0.05). CBNP using ratio 1:4 also has highest percentage followed by 1:2 and 1:3 which were 42.05 ± 5.68%, 41.28± 8.91%, and 36.60 ± 7.56% respectively.

Solubility and Stability

To determine the solubility of C. asiaticacrude chloroform extracts before and following the encapsulation process, C. asiaticacrude chloroform extracts and CBNP were dissolved in water. The results were interpreted by using Randomized Complete Block Design (RCBD) withDuncan’s multiple range tests in SAS program version9.3as showed in table3. The result showed that C. asiaticacrude chloroform extracts has highest solubility in water which is 244.34 ± 17.10µg/ml. But, there are no significant difference between C. asiaticacrude chloroform extracts with others CBNP (p<0.05). While, CBNP using ratio 1:4 has lowest solubility which is 168.86 ± 78.78 µg/ml. The process of making CBNP improve hydrophobic capacity of crude extract. It is difficult for water-soluble biological active compounds to enter through cell membrane of both human and pathogenic microorganism, which has hydrophobic characteristics. So, improving of hydrophobic capacity of crude extract make it can be better in entering through the cell.

Figure 1: Stability of CBNP in PBS (pH 7.4) at 37˚Cover period of 6 hours. Click here to View Figure

 

To study the stability of CBNP, it was incubated in PBS (0.01M, pH 7.4) and calculate their concentration with time by equation 4. CBNP showed very stable under the condition over period of 6 hours as showed in figures1. The nanoparticles process made the stability of C. asiaticacrude chloroform extracts by protecting it from hydrolysis and biotransformation¹¹. CBNP provided stability of the C. asiatica crude extract so, it can be used as process to prolong shelf-life of plant extract.

Figure 2: Release rate of C. asiatica from CBNP in vitro in PBS (in artificial gastric juice at pH 2.0 and artificial intestinal juice at pH 7.4)at 37˚C over period of 6 hours.   Click here to View Figure

 

Release Kinetic in vitro

In the study of release kinetic in vitro, artificial gastric juice (0.01 M PBS pH 2.0) and artificial intestinal juice (0.01 M PBS pH 7.4) were used to imitate the environment in stomach and intestine. The cumulative percentage of released C. asiaticawere determine as showed in figure2.The release rate of C. asiatica from CBNP using same ratio in the gastric juice was tend to higher than intestinal juice. CBNP using ratio 1:4 released highest in both artificial gastric juice (0.01 M PBS pH 2.0) and artificial intestinal juice (0.01 M PBS pH 7.4). At pH 2.0 the BSA protein was unfolded and release C. asiatica out from the particles.  BSA was most stable at pH 7 but rapidly degraded by aggregation and hydrolysis at pH 2²⁷.CBNP can be release in very low pH so, it can be apply to be oral medicine²⁸.

Conclusions

The different ratio of C. asiatica to BSA (1:2, 1:3, and 1:4) has effect on bioavailability.CBNP were less solubility in water than crude extractthus, CBNP can improve hydrophobic capacity of C. asaiticacrude extract.So, this study is focus on improving ability of C. asaiticacrude extract in absorption to the cell.Higher hydrophobic capacity of the CBNP makes it can penetrate into cell membrane and enter to the cell.These result is parallel to antibacterial activity of CBNP that showed 2 times higher than crude extract. Moreover, CBNP has no significantly different in total phenolic content compare with crude extract, but has significantly lower in FRAP and DPPH (p<0.05). CBNP also showed stability and releasing of C. asiatica in PBS.It can be conclude that CBNP at ratio 1:4 is most effective in an economical way because using less crude extract. The development of CBNP would be a promising improving bioavailability of C. asaiticacrude extract.

Acknowledgements

This research is supported by the Assumption University (research grant P-58-388).

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