Study on Biotransformation of Traditional Chinese Herbal Medicines Complex

Feng Chunbo, Qiao Xiaoling, Cao Ping

Shanghai Jahwa United Co., Ltd., China

Abstract A newly designed enzymolysis-fermentation combined method to dramatically enhance the actives level and skin benefits of traditional Chinese herbs (TCHs) was developed, biotransformation process under optimal reaction conditions can significantly transform molecular structures and obtained fermentation extracts can deliver better skin benefits on anti-aging, hydration and whitening. Analytical results showed that the ginsenoside Rg3, total sugar, polyphenols and flavonoids in fermented extract were 2～4 times higher than unfermented extract. In-vitro tests including DPPH radical scavenging activity, tyrosinase inhibition, cells proliferation and Hyaluronan-CD44 activity, have showed significant enhancement of efficacy, inhibition rate on DPPH antioxidation as example achieved over 60% improvement, which makes traditional Chinese herbs application more feasible. It is inferred that this study potentially enables the herbs to deliver required bioefficacy in cosmetic application.

Key words enzymolysis; fermentation; biotransformation; traditional Chinese herbs

Introduction

TCHs generally contain polyphenols, flavonoids,polysaccharides and other active components which have antioxidant, antitumor, antibacterial, and sedative effects,and showed considerable high nutritional value and medicinal values.[1]

Recent years, TCHs were also influenced by modern Western medicine and technology. Fermentation or enzymolysis biotransformation of TCHs can enhance their effectiveness and reduce their toxicity, improve the bioavailability and so on, because both biotransformation techniques can produce new components and break down or convert certain common substrate components into compatible components. Furthermore, they can typically increase physiological and biochemical activities of biological substrates by modifying their naturally occurring molecules.[2,3]So studying and exploitation on TCHs with these techniques will bring new and blooming field for extensive application of Chinese herbal medicines.

The aim of this study was to combine both enzymolysis and fermentation methods together at first time to optimize the efficacy and active level of a TCHs complexutilizing enzyme and microorganism to screen and better understand the key features of the art of TCHs, such as unknown interactions between various ingredients and complex interactive biological systems,has so far missed.

Experimental methods

Preparation of traditional Chinese herbal extract

To prepare the extraction of TCHs, herbs were selected according to prescription principles and followed the proportion in Table 1 and were thoroughly grinded by using a laboratory mill to enhance the homogeneity of the sample and then passing it through a 60 mesh sieve.1.0 kg of herbs powder was suspended in 10.0kg water and boiled under stir for 3 h, the herbs residue was boiled with another 10.0 kg water for 1 h after filtration. After cooling, the obtained extract was mixed and reconstituted in EtOH with the concentration of 90% wt over 12 h, then filtrated and condensed under reduced pressure until 1.0 kg extract sample remained. The extract was labeled TCHs Ex and stored at -18℃ until further use.

Table 1. Composition of TCHs Complex

Enzymolysis of TCHs Ex

Pectinase, cellulase and neutral protease have different enzyme activities, therefore, the same enzyme activity(75,000 U) was used to identify the most effective enzyme.0.3 kg of TCHs Ex was taken in enzymatic reactor, and enzymes were diluted with 0.7 kg water first and entered into the reactor, the concentration of enzyme solution was changed relying on the active level, which is primary component working on skin benefits. Enzymolysis experiment was performed at 60℃ for 24 hours and pH of each enzyme was adjusted to maximize their activity.The crude enzyme sample was further treated at 80℃ for enzyme inactivation and filtered through filter paper, and stored at -18℃ until further use (named TCHs-E).

Fermentation of TCHs-E

Five common used microorganism including Saccharomyces cerevisiae, Lactobacillus bulgaricus,Lactobacillus brevis, Lactobacillus sakei, Bacillus subtilis were investigated and incubated in 200 mL of modified culture medium for reactivation. The optimum fermentation conditions were studied and confirmed, that the fermentation experiment was conducted in a 5 L fermentor at 37℃, initial pH value of 6.5±0.5, 100 rpm agitation and an aeration rate of 1 L/min of air for controlling dissolved oxygen for five different bacterial strains, respectively. The culture medium used for fermentation consists of 5.0%glucose and 10.0% tryptone providing growth conditions for microorganism, and the fermentation was performed following the composition of each formula listed in Table 2.

Finally, the 15 fermentation solutions of TCHs were harvested at different points in time (3 d, 5 d and 7 d) and filtered through filter paper to obtain the TCHs sample liquids, after sterilization at 65℃, producing final samples(labeled TCHs-F) for the following experiments.

Analysis of total sugars, polyphenols, flavonoids and ginsenoside Rg3

Total sugar content was determined by the phenolsulfuric acid reaction,[4]using galactose as the standard.The total polyphenol content was measured by the Folin-Cocialteu method,[5]and the total flavonoid content was measured using a colorimetric assay using rutin as the standard.[6]Ginsenoside Rg3 was analyzed using a HPLC-based technique developed by Lee et al.[7]equipped with a quaternary solvent delivery system, an autosampler, and UV detector, with measurements at 203 nm.

Biological activity assessment

1. DPPH free radical scavenging activity

The DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity was determined according to the method adopted by Kriengsak et al. with some modifications.[8]The sample solutions at different concentration were prepared with water, and 2.0 mL of the solutions was sucked to mix with 2.0 mL DPPH solution (0.2 mmol/L), the absorbance of each mixturewas measured three times at wavelength = 517 nm after 40 minutes' reaction at 37℃. The blank solution was absolute ethyl alcohol. The DPPH radical scavenging activity was calculated using the following equation:

Table 2. Fermentation formulas composition with different microorganism

Where,Acis the absorbance of the undetermined sample;Asis the absorbance of the blank solution.

2. Measurement of tyrosinase inhibition

A 1 mL herbs extract or fermented extracts were diluted with 0.9 mL pH6.8 phosphate buffer (K2HPO48.71 g/500 mL + KH2PO46.805 g/500 mL) in the tubes,followed by the addition of 300 mg/L L-tyrosine (1 mL),and finally, 350 units per milliliter of tyrosinase solution(0.1 mL); This reaction solution was mixed by vortexing and incubated for 25 min at 25℃, the absorbance of the reaction solution was measured at 475 nm. The inhibitory effects of the test samples were expressed as percentage of tyrosinase inhibition, as follows:

Where A= absorbance at 475 nm without the test samples (control); B=absorbance at 475 nm without the test samples and tyrosinase (blank); C=absorbance at 475 nm with the test samples and tyrosinase; D=absorbance at 475 nm without the tyrosinase.

3. Determination of cell proliferation

MTT Assay was used to measure cell proliferation.Cells were seeded at a concentration of 5 × 103cells/well in a 96-well plate and grown in DMEM medium containing 10% FBS for 24 h. then cells were starved overnight and were preincubated with the test samples (10%, 20% and 30% of TCHs Ex and TCHs-F ) and vehicle DMSO for 30 min before stimulation with TGF-β1 (10 ng/mL) for indicated times. At the end of the each cultural time point,cells were incubated with 5 mg/mL MTT in phosphatebuffered saline (PBS) for 4 h at 37℃ in 5% CO2. Then 150 μL dissolving reagent DMSO was added to dissolve the formazan crystals. The optical density (OD) was determined using ELISA plate reader with a reference wave length of 490 nm.

4. Measurement of Hyaluronan-CD44 expression

Pretreated cells with the test samples and blank were added to the anti-CD44 antibody and incubated at 4℃ for 12 h, and washed off with PBS for 3 times (3 min/time).The cells reacted with fluorescent-conjugated antibody at 25℃ for 2 h, and washed off with PBS again. Fluorescence microscope was used to observe the Hyaluronan-CD44 expression after adding DAPI staining. Green fluorescent stain area shown Hyaluronan-CD44 expression, and cell nucleus presented blue dots.

Results and discussion

Some microorganism and enzymes, for example,Lactobacillus bulgaricus and pectinase, may have the potential of producing new ingredients or reducing the cytotoxicity of herb extracts by fermentation or enzymolysis.[9]Designed TCHs complex aim to remedy various skin issues, including anti-aging (antioxidant activities), whitening (tyrosinase inhibition), and hydration (Hyaluronan-CD44 expression), so complex should contains the ingredients targeting these skin problems, such as ginsenoside, flavonoid, polysaccharides and so on. They are also chosen as tracking markers to monitor the changes of TCHs Ex through enzymolysis and fermentation treatment process, also decided the optimum conditions for this bioconversion method.

Optimization of experimental conditions

TCHs Ex was obtained as contrast group first, then using it mixed with different concentration of enzymes(Table 3) to force the composition changing of active ingredients. The results shown in Table 3 indicated that most of data were greater than the correspondingdata of the TCHs Ex for pectinase treatment, and 1.5%concentration had the greatest effect to produce more Rg3, polyphenols and flavonoids, which proved that enzymolysis with pectinase can induce biotransformation of large to smaller molecules.[7]In contrast, the effect of cellulose and neutral protease was generally worse than that of pectinase, the latter favored more actives formation. So pectinase at 1.50% was used in the followup experiment.

Table 3. Actives content with different concentration of enzyme

As shown in Table 4, under different designed formulas for fermentation process, actives content were significantly changed, lactobacillus have the greater positive influence on the increasing of the Rg3, total sugar and flavonoids. The results are consistent with that reported by literatures.[6-7]Otherwise, many enzymes during the fermentation process can be secreted, which catalyzes the hydrolysis of higher molecular weight of polymers with similar, even different structures.Eventually, formula B showed better fermentation results under all cultured conditions.

Optimized fermentation formula is formula B with lactobacillus bulgaricus that can dramatically increase 320% content of Rg3, 440% content of total sugar, and 257% of flavonoids content after fermentation comparing to the data TCHs Ex. To enhance our understanding of actives content in unfermented extract and fermented extracts, respectively, if it can represent holistic skin benefits or not, in-vitro experiments were conducted to compare the inhibitory effect of unfermented extract and before and after fermentation respectively on DPPH antioxidation,fibroblast proliferation and Hyaluronan receptor.

In-vitro experiments to evaluate the skin benefits

DPPH is a stable free radical that accepts an electron or a hydrogen radical to become a stable diamagnetic molecule. When DPPH encounters a proton-donating substance, the radical is scavenged and the absorbance at 517 nm is reduced. Therefore, DPPH is often used as a substrate to evaluate the antioxidant activity, which linked with anti-aging skin benefits. The data shown in Table 5 that the DPPH radical scavenging activity of the fermented sample significantly increased referring to TCHs Ex which is consistent with the high level of Rg3 and flavonoids.

Table 5. In-vitro experiments results

The efficacy of fermented extract in inhibiting tyrosinase were investigated comparing to unfermented extract as well, the results shown in Table 5 that there is not significantly enhancement observed, TCHs-F formula B only delivered 16% increasing, that might be linked with the same level of polyphenols content.

In the process of skin aging, expression of MMPs increases, causing more collagen degradation and resulting in aged skin with wrinkle and skin laxity. This study examined the abilities of fermented extract and unfermented extract to improve the TGF-β1content which is well known to promote collagen synthesis through a series of conversion process within the cells.Compared with unfermented extract, fermented extract shows greater potential of growth and cell proliferation listed in Table 5, it is interesting to note that formula B with higher Rg3, sugar and flavonoids content still deliver the high proliferation benefit.

Table 4. Actives content under different fermentation formulas

Figure 1. Hyaluronan-CD44 expression with different extracts

HA plays an important role in CD44-mediated keratinocyte activity and epidermal aging functions, and CD44 can generate HA expression and promote its production.Figure 1 depicted that TCHs-F formulaB has a better promotion of CD44 expression comparing TCHs Ex as well.

Our results revealed that the presence of Rg3, total sugar,polyphenols and flavonoids led to high antioxidant activity,good tyrosinase inhibition rate, great cell proliferation and CD44 expression activities. Based on these observations, the fermented extracts likely exhibit better skin benefits.

Conclusion

It can be concluded that by using the newly designed enzymolysis and fermentation biotransformation process under optimized conditions, which can transform the water extract to fermented extracts with higher actives level and better skin benefits. Analytical testing results showed that the actives content in fermented TCHs was dramatically increased to 2～4 times higher than unfermented TCHs. Biotransformation process can enhance the release of functional ingredients from the extracts. Moreover, in-vitro tests including DPPH radical scavenging activity, cells proliferation and Hyaluronan-CD44 receptor, have showed significant enhancement of efficacy, inhibition rate on DPPH antioxidation as example achieved over 60% improvement, which makes TCHs application more feasible. It is inferred that this study potentially enables the TCHs to deliver required bio-efficacy in cosmetic application.