WORLD SCIENCE AND TECHNOLOGY

MODERNIZATION OF TRADITIONAL CHINESE MEDICINE AND MATERIA MEDICA

Volume 12, Issue 2, April 2010
Online English edition of the Chinese language journal

Cite this article as: Mode Tradit Chin Med Mater Med, 2010, 12(2): 150–154 REVIEW

Biotransformation of Chinese Herbs and

Their Ingredients
Ma Baiping*, Feng Bing, Huang Hongzhi, Cong Yuwen
Beijing Institute of Radiation Medicine, Beijing 100850, China

Abstract: Biotransformation has been extensively applied in herbal fermentation processing, metabolism study of traditional Chinese med-
icine (TCM), structural modification and synthesis of active components of TCM. Selecting steroidal saponins as the subject, the selective
hydrolysis of glycosyl residues, glycosylation of molecules, and the modification of their aglycone were systematically studied by biotech-
nology in this work. Some active microorganisms were screened, several specific enzymes were purified, and their substrate specificities
and transformed regularities were summarized. These results lay a better foundation for determining the key core of the active components,
specifically modifying their structures, constructing the focused library with the structural diversity, and manufacturing some new drug
candidates which are rare in herbs. They may also provide a kind of valuable strategy and approach support for herbal substance library
construction as well as the active ingredients research of Herbalome Project.

Key Words: Traditional Chinese medicine, steroidal saponin, biotransformation, glycosidase, structural modification

Biotransformation is a biochemical reaction to modify the
structure of the xenobiotics by vegetal cellular or organ, an-
imal cellular, microorganism and its organelle, and isolated
enzyme, which is mainly enzyme-catalyzed reaction. Advan-
tages often associated with biotransformation are pronounced
exquisite chemoselectivity, regioselectivity, stereoselectivity,
less by-product, and easy to operate under mild conditions.
Besides the advantages mentioned earlier, biotransformation
by means of organism or enzyme as biocatalyst has been wi-
dely applied in traditional Chinese medicine (TCM) and their
active ingredients for modifying and producing biologically
active metabolites. This technology mainly appears in (i) Her-
bal fermentation processing, which is to produce bulk secon-
dary metabolites from domestic plant by metabolic mechanism
of herb itself (microorganism or plant cell). The fermentation
technologies of Semen Sojae Praeparatum (Dandouchi), Massa
Medicata Fermentata (Liushenqu), Rhizoma Pinelliae Ferm-
entata (Banxiaqu), and Mass Galla chinesis et camelliae Fer-
mentata (Baiyaojian) have been embodied in Chinese Pharm-
acopeia 2005 edition [1]. Hongqu, for example, is a fermenta-
tion preparation which is fermented on rice by Monascus pur-
pureus W. (Hongqumei), and it has been used as both food
and medicine in China for more than 1000 years. This prepar-
ation is found to have various bioactive ingredients with lo-
wer hyperpiesia, lower hypercholesterol, lower hyperglycemia,
antimicrobial, and antitumor properties. Many relevant bio-
active ingredients have been isolated from Hongqu, such as
monacolins, monascorubin, ergosterin, and notalin [2]. The di-
versity of microorganisms provides extensive strains with va-
rious properties, which might act with different effectiveness
on fermentation processing [3]. (ii) To build the microbial
model for metabolic mechanism of TCM. Based on the simi-
larity of metabolic mechanism between microorganism and
human, simulating the metabolic mechanism of TCM by mi-
croorganisms contributes to our understanding of the relevant
pharmaceutical metabolites in human. For example, soy iso-
flavones was transformed into corresponding aglycone and
equol by human intestinal microflora; peoniflorin took effect
only when it was converted to corresponding aglycone, pae-
onimetabolin [4]. For the past few years, with further studies
on microbial transformation, many kinds of microorganisms
are found to metabolize a variety of xenobiotics in region- and
stereo-selective manners that are similar to those in mam-
malian enzyme systems (cytochrome P450 enzymes and glu-
curonidases), such as hydroxylation, acetylation, N-dealkyla-
tion, and β-glucuronic acid conjugate reaction containing both
phase I (oxidative) and phase II (conjugative) biotransforma-
tion mechanisms. Among them, Cunninghamella species has
been used extensively as microbial model to mimic mamma-
lian hepatic metabolism of aromatic xenobiotics [5]. Many
studies on building microbial models of TCM have also been
reported in China. For example, the model of in vitro glyco-
side productions by the enzyme systems produced fermenta-
tion procedure of microorganisms has been set up to obtain
their corresponding aglycones that can enhance the bioavail-
ability of active ingredients [6, 7]. (iii) To synthesize and mo-

Received date: 12 October 2009

* Corresponding author. Tel: +86-10-66930265; E-mail: ma_bp@sohu.com
Foundation item: Supported by the General Program of National Natural Science Foundation of China (30572333, 20672142, 30973632), Major Program of
Municipal Natural Science Foundation of Beijing (7090001), and China Postdoctoral Science Foundation (20070410510).
Copyright © 2010, World Science and Technology Press. Published by Elsevier BV. All rights reserved.
 Ma Baiping et al. / Mode Tradit Chin Med Mater Med, 2010, 12(2): 150–154
dify the active ingredients of TCM, broaden the library of na-
tural products, and design the pathways that synthesized drugs.
For example, Gastrodine was synthesized by microorganisms
or cell suspension culture of plant [8, 9]. Ginsenosides Rg3
and C-K, which are rare ginsenosides in nature, were produced
by microorganisms and enzymes [10]. Camptothecin was trans-
formed to 10-hydroxycamptothecin [11]. Recently, the app-
roach of directly prepare sapogenin (diosgenin) from Diosc-
orea zingiberensis C.H. Wright by microorganisms has ob-
tained interesting development. The rare ingredients with na-
tural pharmaceutical activity in nature can be synthesized com-
pletely by biotechnology. For instance, bilobalide was synthe-
sized by several key enzymes. among those enzymes, the ge-
nes of six key enzymes, which were the enzymes synthesiz-
ing the precursor of bilobalide, were identified and cloned.
These studies provide the synthesis pathway of bilobalide in
molecular level by metabolic engineering technique, and the
essential functional groups and the target point catalyzed by
enzymes for enhancing the synthetic efficacy [12].
Steroidal saponins are a kind of important active compounds
with various relevant pharmaceutical activities such as anti-
dementia, platelet aggregation, antitumor, immunoregulatory,
and antibacterial activity [13-15]. Due to structural complex-
ity, it is so difficult to synthesize or modify steroidal saponins
by chemical approach, especially their oligosaccharide of su-
gar chain. However, producing steroidal saponins with higher
activity and lower toxicity by biotechnology is a tendency. We
have also conducted some studies on steroidal saponins using
the technique of biotransformation.
1 Hydrolyzing selectively the glycosyl moiety in
steroidal saponins
A novel enzyme from Curvularia lunata was found to be
capable of selectively hydrolyzing the terminal α-1, 2-rham-
nosyl residues at C3-sugar chain of steroidal saponins accord-
ing to the obtained results from 28 steroidal saponins and 6
triterpenoid saponins tested. A total of 18 secondary saponins
without terminal α-1, 2-rhamnosyl residues were isolated and
identified based on the data of spectra. And the specific enzy-
matic regularity of hydrolysis for steroidal saponins was sum-
marized as follows: the enzyme had the strict selectivity for
Fig. 1 The substrate specificity of Curvularia lunata in steroidal saponins
type of glycosyl group and its linked site (α-1, 2-rhamnosyl
residues), and it had no strict selectivity for aglycones, the
number of glycons in sugar chains, and the branched chains
or the linear chains. The enzyme had no activity for the rha-
mnosyl group of triterpenoid saponin (Fig. 1) [16, 17]. Kler-
zyme-150 enzyme, an enzyme preparation, was found to be
able to selectively hydrolyze the terminal α-1, 4-glycosyl re-
sidues at C3-sugar chain of steroidal saponins based on the
obtained results from 29 steroidal saponins tested. A total of
26 secondary saponins hydrolyzed α-1, 4-glycosyl residues
were identified when compared with reference standards by
using thin-layer chromatography and high performance liquid
chromatography analyses. The specific enzymatic regularity
of hydrolysis for steroidal saponins was summarized as fol-
lows: the enzyme had the strict selectivity for the linked site
of glycosyl group (α-1, 4-glycosyl residues), and it had no st-
rict selectivity for aglycones, the number of glycons in sugar
chains, and the branched chains or the linear chains (Fig. 2).
These studies provide a number of important information for
structural modification of steroidal saponins of our interest
and we understood the role and significance of rhamnosyl gr-
oup in relevant pharmaceutical activities. It is also found that
the glucosyl residue at C3-sugar chain of steroidal saponins
hardly be hydrolyzed by some enzymes preparations such as
β-glucosidase, amylase, and cellulase. However, an approach
of microorganism in mixed culture was used to hydrolyze the
glucosyl residue at C3-sugar chain of timosaponin AIII and
timosaponin BII. The results showed that the glucosyl resi-
dues of these substrates can be hydrolyzed by three microor-
ganisms in mixed culture with high yield, although they can-
not be hydrolyzed by any individual microorganism mentioned
earlier in pure culture. The catalytic mechanism of three micro-
organisms in mixed culture was systematically investigated.
Two enzymes which hydrolyze, respectively, the β-glucuronic
acids of glycyrrhizin (GL) and baicalin were purified from an
enzyme preparation, and their substrate specificities presented
that the enzyme hydrolyzing the β-glucuronic acids of GL
cannot hydrolyze the β-glucuronic acids of baicalin, and vice
versa. The two enzymes appeared much different in substrate
specificity.
 Ma Baiping et al. / Mode Tradit Chin Med Mater Med, 2010, 12(2): 150–154

Fig. 2 The substrate specificity of Klerzyme-150 in steroidal saponins

R1=Glc, Gal; R2=Glc, H; R3=Glc, H; R4=OH, H

R5=Glc (1, 4), Glc (1, 4)Glc (1, 4); R6=Glc (1, 4), Glc (1, 4)Glc (1, 4)

R1=Glc, Gal; R2=Rha (1, 2), H; R3=OH, H; R4=Glc (1, 3), Glc (1, 3)Glc (1, 4)

Fig. 3 The substrate specificity of CGTase in steroidal saponins

2 Glycosylation of steroidal saponins
Cyclodextrin glucosyltransferase (CGTase) was found to be
able to transfer the glucoses (donor) from cyclodextrin, dex-
trin, and starch to the glucosyl groups (acceptor) of steroidal
saponins. Five spirostanosides and one furostanoside were ca-
talyzed into their corresponding glucosylated derivatives, and
19 derivatives were isolated and elucidated based on the data
of spectra (Fig. 3). The specificity of the enzyme for glucosy-
lation in steroidal saponin showed that CGTase was capable
of glucosylating steroidal saponins with additional α-D-glu-
cosyl groups, and the β-D-glucosyl group in steroidal sapon-
ins was the only acceptor for glucosylation. However, whether
a β-D-glucosyl-acceptor to be glucosylated depended strongly
on the environment in which it located. The specificity of the
enzyme for glucosylation in steroidal saponin was summa-
rized as follows: (i) if the C2-OH, C3-OH, and C4-OH of β-
D-glucosyl-acceptor were not replaced by any glycosyl group,
the additional glucoses were attached to the C4-OH of acce-
ptor to give the derivative with additional α-D-1, 4-glucosyl
group. (ii) If the C2-OH of the β-D-glucosyl-acceptor was oc-
cupied by a glycosyl group such as 1, 2-glycosyl group, the
first additional glucose was linked to the C3-OH of acceptor
to obtain the derivative with additional α-D-1, 3-glucosyl gr-
oup, while the second additional glucose was linked to the
C4-OH of the first additional glucose to give the derivative
with α-D-1, 3-glucosyl→ α-D-1, 4-glucosyl group. (iii) If the
C3-OH and C4-OH of β-D-glucosyl-acceptor were replaced
by other glycosyl groups, then this glucosyl group cannot be
an acceptor for glycosylation by CGTase. The characteristics
 Ma Baiping et al. / Mode Tradit Chin Med Mater Med, 2010, 12(2): 150–154
of the enzyme on glucosylation showed that the enzyme had
the higher thermostability with 90°C reaction condition and
higher tolerance in organic solvent with 70% methanol.
3 Modification of aglycon
The enzymatic system from Cunninghamellu elegans was
demonstrated to perform hydroxylation in sarsasapogenin (a
kind of aglycon) to obtain two derivatives. The enzymatic sys-
tem from microorganisms in mixed culture was able to iso-
merize the C22 at sarsasapogenin moiety with β-configuration
resulted from removing the C26-O-glucosyl group of furo-
stanol saponin, which exists rarely in nature.
4 Isolation and elucidation of active enzymes
for steroidal saponins
In order to investigate and reveal the structure–activity re-
lationship and the catalytic mechanism of their active enzy-
mes, three enzymes mentioned earlier were purified after iso-
lation and purification by column chromatography as homo-
geneity, respectively. Their protein fragments were analyzed
by MALDI-TOF/TOF™ proteomics analyzer that indicated,
respectively, these enzymes to be 1, 4-alpha-D-glucan gluco-
hydrolase (EC 3.2.1.3, GA) with the specific property of hy-
drolyzing α-1, 2-rhamnosyl group [18], rhamnogalacturonan
lyase A (EC 4.2.2-, RG-lyase) with the specific property of
hydrolyzing α-1, 4-glycosyl group, and cyclodextrin-glycos-
yltransferase (EC 2.4.1.19) with the specific property of add-
ing α-D-glucosyl group to steroidal saponins.
5 Relevant pharmaceutical activities between
original saponin and secondary saponin
The relevant pharmaceutical activities were screened by us-
ing the original steroidal saponins and secondary steroidal sa-
ponins without α-1, 2-rhamnosyl group as samples, and it is
found and confirmed that (i) Cytotoxicity: The α-1, 2-rham-
nosyl residue at C-3 position of steroidal saponins was the key
group to the cytotoxicity. Compared with their original sapo-
nins, the cytotoxicity of secondary saponins obviously decr-
eased or disappeared. (ii) Inducing platelet aggregation: the
number and type of glycosyl groups in saponins’ sugar chains
play a key role in inducing platelet aggregation or inhibiting
platelet aggregation. The C3-sugar chain of steroidal saponin
is essential for platelet aggregation. The number and type of
glycosyl groups strongly influenced platelet aggregation. A
structure–activity assay has demonstrated that the inducing
platelet aggregation activity was reversed by the derivatives
with α-1, 2-rhamnosyl residue removed. For diosgenin glyco-
sides with triglycosides, the inducing platelet aggregation ac-
tivity of saponin with terminal α-1, 4-arabinosyl group was
much higher than that of saponin with terminal α-1, 4-rham-
nosyl group. For pennogenin glycosides, extending sugar units
from diglycosides to triglycoside, and then to tetraglycosides,
also significantly enhanced their activities. (iii) Inhibiting pl-
atelet aggregation: the steroidal saponins without α-1, 2-rha-
mnosyl residues exhibited the obvious inhibiting platelet ag-
gregation activities. Among them, diglycosides displayed the
most activity. For diosgenin glycosides and pennogenin gly-
cosides, extending sugar units obviously decreased their acti-
vities. The diglycosides exhibited the most inhibiting platelet
aggregation activity [19, 20].
In addition, biotransformation will be also applied in other
fields.
5.1 Study on the active center of TCM ingredients
We have understood the role of α-1, 2-rhamnosyl residue
of steroidal saponins in pharmaceutical activity by compar-
ing original steroidal saponins with their secondary sapon-
ins without α-1, 2-rhamnosyl residues. Timosaponin AIII
(C22-α-configuration) exhibited a favorable inhibiting platelet
aggregation in vitro. However, its isomer with C22-β-con-
figuration, which was prepared by microorganism in mixed
culture, did not exhibit any activity. The results suggested th-
at C22-α-configuration was the essential structure to inhibit
platelet aggregation.
5.2 Structural modification
With the studies deeply on microorganisms or enzymes and
the find of more specific enzymes, biotransformation makes
it possible for the structural modification of active TCM in-
gredients we desired. A study showed that α-1, 2-rhamnosyl
residue of steroidal saponins was the active fragment of cy-
totoxicity. We can selectively hydrolyze the α-1, 2-rhamnosyl
residues of steroidal saponins by Curvularia lunata to de-
crease the cytotoxicity. Wang Yun et al. [21] reported that a
microorganism was able to specifically hydrolyze the glu-
curonosyl moiety of GL to obtain GAMG with a glucuronosyl
moiety removed. The sugariness of generated GAMG was
1000 times higher than sucrose, and five times higher than
GL.
5.3 Combinatorial biocatalysis
Combinatorial chemistry is a powerful strategy and method
for drug discovery. When biocatalysis is brought to the field
of combinatorial chemistry, combinatorial biocatalysis more
efficiently generates high-quality libraries we desired by us-
ing enzymes and microorganisms to find higher active and
efficient lead compounds [22]. We have found that one en-
zyme or one microbial strain was able to catalyze one single
compound to obtain several relevant derivatives with higher
cytotocity [23, 24]. In order to enhance the activity and pro-
ductivity of active compounds, reduce the toxic ingredients,
and produce the novel active compounds, the biotransform-
ation process can exploit the comprehensive harmonies of
various chemical compositions and also biosynthesize novel
chemical components by modifying the molecular structures
 Ma Baiping et al. / Mode Tradit Chin Med Mater Med, 2010, 12(2): 150–154
of the ingredients from TCM to build the focused library
[1].
5.4 Manufacturing new drug candidates for
development
TCM is often taken orally, and metabolized by intestinal mi-
croflora and enzymes in intestines to generate a series of met-
abolites, and then absorbed into blood to act [25]. However,
these metabolites with potential activities are often minor or
rare ingredients in TCM. The biotransformation is capable of
producing the minor or rare derivatives in bulk for studying
their efficacy and safety, and developing as new drug such as
ginsenoside CK [26], resveratrol (prepared from Rhizoma
References
[1] Tang YJ, Xu XL, Li Y et al. Simultaneous biotransformation
of various chemical compositions from traditional Chinese
medicines. Chin J Nat Med 2007; 5(4): 241-244.
[2] Tu ZY, Shao W. Current application and trend of red koji in
the world. China Brewing 2008; 7: 7-9.
[3] Liu LJ, Pan Y. A approach to Fermentation Processing of
TCMs. Research and Practice of Chinese Medicines 2009;
23(1): 72-76.
[4] Yuan JP, Wang JH, Liu X. Metabolism of dietary soy isofla-
vones to equol by human intestinal microflora--implications
for health. Mol Nutr Food Res 2007; 51: 765-781.
[5] Asha S, Vidyanathi M. Cunninghamella-A microbial model
for drug metabolism studies-A review. Biotechnology Advances
2009; 27: 16-29.
[6] Xu MM, Wang JF, Xu C. Study on Microbial Transformation
of Fructus Arctii World Science and Technology/Moderni-
zation of Traditional Chinese Medicine and Material Medica
2006; 8(2): 4-7.
[7] Qin S, Zhou CL. Application of Microbial Transformation in
Medicine Metabolization Mode in vitro by Cunninghammella
Matruchot. Strait Pharmaceutical Journal 2004; 16(1): 4-8.
[8] Dai JG, Gong Z, Zhu DM et al. Biotransformation of Gas-
trodin by Cell Suspension Cultures of Catharanthus roseus.
Acta Botanica Sinic, 2002; 44(3): 377-378.
[9] Zhan JX, Guo HZ, Dai JG et al. Biotransformation of Gastrodin
by Mucor spinosus. Journal of Chinese Pharmaceutical Sciences
2001; 10(4): 187-189.
[10] Zhang YX, Chen XY, Zhao WQ. Advances in studies on
biotransformation of ginsenosides. Journal of Shenyang Pha-
rmaceutical University 2008; 25(5): 410-422.
[11] Wang WL, Huang XR, Zhang LP. Advances in studies on
cell culture of medicinal plant. Prac J Med & Pharm. 2007;
24(7): 867-869.
[12] Liu WH, Chen M, Liao ZH et al. Advances in studies on
biosynthetic pathway and biotechnology of ginkgolides. Chi-
nese Traditional and Herbal Drugs 2007; 38(6): 941-945.
[13] Deng Y, Ma BP, Cong YW et al. Research progress in im-
proving brain function by Anemarrhena asphodeloides Beg
Polygoni Cuspidati) [27], and genistein [28].
With the development of gene engineering and protein en-
gineering, these techniques provide powerful supporting for
studying the biotransformation, particularly for understanding
the regularity of microbial catalyzed, for finding the specific
enzymes, and also for developing TCM products. Recently, Her-
balome pronounced a project about the systematic research
and development of TCM (Herbalome Project). Biotransfor-
mation might also provide a valuable strategy and approach
for the herbal substance library construction as well as the her-
bal active ingredients research of Herbalome Project.
and its effective components. Chin Pharmacol Bull 2008; 4
(5): 576-579.
[14] Li SY, Zhao ZH, Pei HY et al. Effect of timosaponin-AIII on
formation of thrombus. Bull Acad Mil Med Sci 2006; 30(4):
340-342.
[15] Deng Y, Ma BP, Cong YW et al. Protective effects of timo-
saponin BII on primary neurons against beta amyloid peptide
25-35. Chin Pharmacol Bull 2009; 25(2): 244-247.
[16] Feng B, Ma BP, Kang LP et al. The microbiological trans-
formation of steroidal saponins by Curvularia lunata. Tetra-
hedron 2005; 61: 11758-11763.
[17] Feng B, Kang LP, Ma BP et al. The Substrate Specificity of a
Glucoamylase with Steroidal Saponin-rhamnosidase Activity
from Curvularia lunata. Tetrahedron 2007; 63: 6796–6812.
[18] Feng B, Hu W, Ma BP et al. Purification, Characterization,
and Substrate Specificity of Glucoamylase with Steroidal sa-
ponin-rhamnosidase Activity from Curvularia lunata. Applied
Microbiology and Biotechnology 2007; 76: 1329-1338.
[19] Fu YL, Yu ZY, Tang XM et al. Pennogenin glycosides with
spirostanol structure are strong platelet agonists: structural
requirement for activity and mode of platelet agonist syner-
gism. J Thromb Haemost 2008; 6(3): 524-533.
[20] Zhao Y, Kang LP, Liu YX et al. Steroidal saponins from the
Rhizome of Paris polyphylla Smith var. yunnanensis and their
Cytotoxic Activities. Planta Medica 2009; 75: 356-363.
[21] Wang Y. Glycyrrhizin was transformed to mono-β-glucuro-
nide-glycyrrhizin by a microorganism. Science and Tchnology
of Food Industry 2005; 26(11): 151, 160.
[22] Lv QJ, Huang HZ, Ma BP et al. Methodology of New Drug
Research in Pharmacology-Combinotorial Biocatalysis. Che-
mical Industry Press: Beijing 2007; 637-641.
[23] Quan B, Ma BP, Feng B et al. The Microbiological Transfor-
matioin of Protodioscin by Aspergillus oryzae. Chin J Nat
Med. 2006; 4(5): 377-381.
[24] Zhao Y, Feng B, Kang LP et al. Biotransformation of Di-
chotomin by Pectinex BE XXL. Chin J Nat Med. 2009; 7(5):
380-389.
[25] Li YM, Li XM, Zhu Z. Advances in biotransformation of
glycosides from Chinese medicinal by human intestinal bac-
 Ma Baiping et al. / Mode Tradit Chin Med Mater Med, 2010, 12(2): 150–154

teria. World Chinese Journal of Digestology 2008; 16(19):
2144-2148.
[26] Wang L, Yu XX, Yu HS et al. Transformation of protopana-
xadiol saponin to compound-K. Journal of Dalian Polytechnic
University 2008; 27(1): 22-25.
[27] You S, You K, Wang X et al. Extraction of piceid and its bio-
transformation. Journal of Shenyang Pharmaceitical Uni-
versity 2009; 26(4): 312-315.
[28] Jin FX, Yu HS, Zhang CZ et al. Biotransformation of Natural
Products. Chemical Industry Press: Beijing 2009; 234-239.