Belamcandae chinensis rhizoma – a review of phytochemistry and bioactivity

Dorota Woźniak, Adam Matkowski

PII: S0367-326X(15)30074-5
DOI: doi: 10.1016/j.fitote.2015.08.015
Reference: FITOTE 3253

To appear in: Fitoterapia

Received date: 6 July 2015

Revised date: 28 August 2015
Accepted date: 30 August 2015

Please cite this article as: Dorota Woźniak, Adam Matkowski, Belamcandae chinen-
sis rhizoma – a review of phytochemistry and bioactivity, Fitoterapia (2015), doi:
10.1016/j.fitote.2015.08.015

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 ACCEPTED MANUSCRIPT
Belamcandae chinensis rhizoma – a review of phytochemistry and bioactivity

Dorota Woźniak* & Adam Matkowski

*corresponding author

Department of Pharmaceutical Biology and Botany. Medical University of Wroclaw, ul. Borowska 211, PL-

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50556 Wroclaw, Poland.

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tel: +48 717840497

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fax: +48 71 7840498

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e-mail: pharmaceutical.biology@wp.eu

Abstract NU
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Belamcandae chinensis rhizoma, is a rhizome of Iris domestica (syn. Belamcanda chinensis). Under the Chinese

name she gan, it is extensively used in Traditional Chinese Medicine and other East Asian phytotherapy
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systems. Recently, the monograph of Belamcandae chinensis rhizoma has been included in the European
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Pharmacopoeia. This review provides a comprehensive summary and systematizes the literature data on

ethnobotanical uses, chemical constituents and biological effects of Belamcandae chinensis rhizoma and its
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components.
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The main group of phytochemicals identified in the dried rhizoma are polyphenols such as isoflavones,
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xanthones glycosides, stilbenes, simple phenols and quinones. Another characteristic class of substances are

triterpenopid iridals.

The most typical traditional usage of Belamcandae chinensis rhizoma is for healing respiratory diseases but

most of pharmacological research so far has been focused on isoflavones and their estrogenic properties. In

pharmacological research, it has been mainly considered as a source of tectorigenin – a phytoestrogene with

therapeutic potential in hormone-dependent cancer. The most active isoflavones are tectoridin, tectorigenin and

irigenin. The available literature indicates that Belamcandae chinensis rhizoma can prevent excessive oxidation

of biomolecules based on various antioxidant mechanisms: transition metal ions reduction, inhibition of lipid

peroxidation, free radicals scavenging. The other biological activities proven by a number of in vitro studies

include: anti-mutagenic, anti-inflammatory, anti-angiogenic, hypoglycemic.

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In conclusion, the knowledge about Belamcandae chinensis rhizoma has been growing rapidly in the recent

years, but there are still significant gaps in our understanding of its bioactivity, therapeutic value, and roles

played by each of the numerous phytochemicals.

Key Words

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Iris domestica; isoflavones; xanthones; iridals; phytoestrogens; she gan.

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1. Introduction

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A number of indigenous Asian medicinal plants have become popular worldwide throughout centuries of

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traveling, trade, and intercultural communication. Some of the drugs such as rhubarb and ginseng to name just

the most recognized, have been known and valued in Western civilizations for a long time. Medicinal plants are

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an essential part of Traditional Chinese Medicine, an ancient complex therapy considered today as one of the

most complete complementary medicine system. Chinese Herbal Medicines included in Chinese Materia
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Medica listings cover more than 1500 plants and thousands more are used as surrogates. A number of herbs and

preparations are available in worldwide pharmacies, herbal stores and even more (and unsupervised) over the
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Internet.
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TCM is a vast resource of information about the herbal drugs and bases on a holistic view of health and disease.

However, this knowledge is incompatible to the evidence-based medicine approach of the modern science. On
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the other hand, according to the current trends in ethnopharmacology and pharmacognosy [see for example the
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Planta Medica special issue on TCM, Vol. 76, 2010], this experience should be understood and used for the
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benefit of all world’s healthcare, which can be achieved by providing a scientific background for the TCM

practice.

Recently, several other East Asian herbs, like Belamcandae chinensis rhizoma, have been included into the

European Pharmacopoeia and many more are on the waiting list [2]. However, some of the fundamental herbs

have not been widely popularized in other parts of the world, and have not yet received a thorough review in the

current literature. One of the best examples to mention is Belamcandae chinensis rhizoma – underground parts

of Iris domestica (L.) Goldblatt & Mabb. (syn. Belamcanda chinensis (L.) DC.). Beside the phytochemical

analytic and preparative work, there are more and more publications about the pharmacological activity,

including the HPLC-guided confirmation of the role of isoflavones in the antidiabetic activity [3]. The boosting

interest in this plant, rather little known outside Asia, should be an additional indication to its therapeutic

potential. Listing in European Pharmacopoeia and growing popularity of TCM worldwide warrants further

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interest in this drug and more research is to be expected. For this reason, we attempt to summarize the state-of-

the art in available phytochemical and pharmacological records of Belamcandae chinensis rhizoma and discuss

it in relation to the traditional indications.

Scientific herbal literature reviews showed that Iris, a genus of about 300 species of in the family Iridaceae,

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indicates a common phytochemical profile of the main groups of constituents: isoflavonoids, stilbens, xanthones

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and triterpenoid iridals. The European species of Iris have not been extensively used in traditional medicine.

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Among the European Iris species, Iris x germanica L comes to mind as being traditionally used for various oral

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and topical applications (used as several varieties and subspecies, for example as Iridis florentinae rhizoma).

Nonetheless, the European Iris species had never reached the status of herbs of such importance as

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Belamcandae chinensis rhizoma in the TCM. Presumably, this is the result of a transitory decline of European

herbalism practice which could be regarded as equivalent to Eastern systems such as Traditional Chinese
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Medicine (TCM) and its wealth of experience with plants. Belamcandae chinensis rhizoma and two other Iris

species are essential drugs of the TCM. Comparing the Iris species, some of them e.g. I. domestica
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(Belamcanda chinensis), I. tectorum, I. dichotoma and I. japonica bear a striking resemblance of phytochemical
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profile but even then they also contain other chemical components which define them as separate drugs. For

instance, I. tectorum (Chuan she gan) can be distinguished from I. domestica (she gan) only by its high
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acetophenone glycosides content and the absence of resveratrol and xanthones [1]. Among the Iris species, only
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the rhizome of I. domestica (under the old name Belamcanda chinensis) is considered as the authentic origin of
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she gan accepted by the Chinese Pharmacopoeia (version 2010). However, literature sources and market

investigations point to rhizomes of two other Iris species – I. tectorum Maxim. and I. dichotoma Pall., which

have been used as the adulterants for Belamcandae chinensis rhizoma both in the past and nowadays.

With respect to this species, there has been a boom in the number of papers in the recent years, with (PubMed,

accessed August 13th, 2015) 28 papers in the last five years compared to the total 78 PubMed records for the

search term “belamcanda” or “belamcandae”. On the other hand, most of the earlier literature was published

only in the Chinese language in local journals.

1.1. Literature search

For a comprehensive literature overview, we analyzed the published phytochemical and pharmacological data
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available through several search engines, such as SciFinder, ISI Web of Science, Scopus, and
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GoogleScholar, ®PubMed, using ‘belamcanda’ ‘belamcandae’ or ‘iris domestica’ as the search keywords. We

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disregarded publications pertaining to agronomy, plant pathology, ecology and other unrelated topics (unless

any phytochemical or pharmacological data were provided in them) as well as those about other species. In a

few cases, we found publications using bibliographical data from the reference lists of newer papers to verify

the cited information. We have also included a few pharmacological publications on some pure compounds

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found in Belamcandae chinensis rhizoma, but obtained from other plants.

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2. Botanical description

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The species used to be commonly known under a botanical name Belamcanda chinensis (L.) DC, as a separate

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monospecific genus native in South-East Asia (Bhutan, China, Japan, Korea, Myanmar, Philippines, Thailand,

Ussuri region of Russia, Vietnam). It has also been naturalized in some parts of Europe and North America as

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an ornamental garden plant. The species has long been understood to be most closely related to Iris, but has

nevertheless been maintained as a separate genus because of its distinctive floral, fruit and shiny-black seeds
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morphology. In some older sources, it has been also known under the botanical names – Pardanthus chinensis,

Ixia chinensis. Recently, upon detailed morphological and molecular investigation B. chinensis has been
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conclusively placed within Iris under a name Iris domestica Goldbl. & Mabb., most closely related to I.
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dichotoma [4]. This classification has been recently challenged [5], so the adventurous history of this species

seems to continue and future re-classifications cannot be excluded. The common English names include:
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blackberry lily, leopard flower or leopard lily.

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It is a perennial herb with sword-shaped, distichous leaves, branched inflorescence reaching 60-120 cm. The
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flowers have six segment perianth, with subequal tepals, their color can range from red or orange to yellow but

typically flowers are orange with darker reddish spots. Plant blooms in summer but single flower is blooming

during one or two days only. The capsule fruit hold black glossy seeds bearing resemblance to a blackberry that

gives the plant its common name blackberry lily. The underground parts of this plant – yellow rhizomes (she

gan, Belamcandae chinensis rhizoma) have been used since centuries as a natural remedy in Traditional Chinese

Medicine (TCM) and some other East Asian indigenous therapy systems.

3. Traditional use

Belamcandae chinensis rhizoma, (pinyin: she gan, Korean: sa kan) is one of the most important Chinese Herbal

Medicines. The TCM characteristics are bitter and cold, related to lungs, with sinking and dispersing action,

descending lung “qi”. Its traditional indications are sore throat - treats sore throat by eliminating heat, toxins and

phlegm, dyspnea, dissolves sputum and relieves wheezing caused by accumulation of heat and phlegm in the

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lung [5-7]. Similar usages of this herb are also practiced in Korean, Vietnamese, Philippino Traditional

Medicines, and Japan’s Kampo medicine. The recommended dosage is 6-10 g in decoction. In the TCM, she-

gan is frequently prescribed in combination with other herbs, for example Mori cortex, Asari herba, Pinelliae

rhizoma, Scutellariae radix, Platycodonis radix, Ephedrae herba, Schisandrae fructus, or even with earthworms

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(Di long), for treating various respiratory disorders [5- 10]. The specific composition of the mixture depends on

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the desired therapeutic effect and the individual TCM diagnosis.

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According to Porter Smith and Stuart [7], who published Chinese Materia Medica at the beginning of the 20th

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century, Belamcandae chinensis rhizoma (under the name Pardanthus chinensis), apart from the aforementioned

respiratory tract indications, has been also used in breast cancer and amenorrhea treatment. This use relates to

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the scientifically verified phytoestrogenic properties. The use of this herb in pregnancy is restricted [6].

In Thai and Vietnamese folk medicine it is also used in menstrual disorders, such as period irregularities,
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amenorrhea or dysmenorrhea [11].

In Nepal, it is used for liver complaints and as expectorant [12].

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4. Phytochemistry
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Belamcandae chinensis rhizoma is rich in a variety of polyphenols from several phytochemical classes:

isoflavonoids, stilbenes, xanthones, and simple phenols [1,3,11,13-31].

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4.1. Phenolic compounds

Flavonoids are the main phenolic compounds of Belamcandae chinensis rhizoma, chemically based upon a
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fifteen-carbon skeleton consisting of two benzene rings A and B linked via a heterocyclic pyrane ring (C). The

position of the benzenoid substituent divides the flavonoids into flavones (2-position) and isoflavones (3-

position). These compounds, obtained from the rhizomes, occur as lipophilic aglycones, more hydrophilic

glycosides or their polymethoxylated derivatives. When glycosides are formed, the glycosidic linkage is located

at the 7 and 4’positions in the isoflavones or in 3-position in flavone glycosides such as astragalin.

Isoflavonoids, the major class of belamcanda phytochemicals are represented by the methoxylated structures

such as: iridin (5,7,3’-trihydroxy-6,4,5- methoxyisoflavone-7-O-glucoside), tectoridin (5,7,4’-trihydroxy-6-

methoxyisoflavone-7-O-glucoside), and their aglycones: irigenin, tectorigenin followed by the less abundant

irisflorentin (albeit, this compound is a quality standard according to PhEur) and iristectorigenin A.

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Structures of predominant isoflavones identified in the Belamcandae rhizoma are presented in Fig. 1.

The isoflavonoid constituents identified so far and their representative structures are shown in Fig. 2, 3, 4

European Pharmacopoeia monograph requires 0.1 % content of irisflorentin in the dried rhizome determined by

reversed phase HPLC (C18 silica column) (Ph Eur 8).

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The five major isoflavone aglycones identified with hyphenated chromatographic techniques LC-MS and LC-

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NMR are tectorigenin, irigenin, iristectorigenin A, irisflorentin, and 5,6,7,3’-tetrahydroxy-4’-methoxyisoflavone

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[12]. These major compounds are accompanied by a number of derivatives differing in their substitution pattern

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and glycosylation. The presence of numerous methoxyisoflavones is also common in other Iris sp. [17,33].

Beside the isoflavones, several flavones are also present: hispidulin, isorhamnetin, rhamnazin, apigenin,

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luteolin, 5,4’-dihydroxy-6,7-methylenodioxy-3’-methoxyflavone and 5,7,4’-trihydroxy-3’,5’-dimethoxyflavone

(Fig. 5) [26,29,33].
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Other characteristic phenolic compounds of this herb belong to the xanthones that give the rhizome its
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characteristic yellow color - mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) and

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isomangiferin (4-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone) , neomangiferin (mangiferin-7-O-β-

glucoside) (Fig. 6 C, D, E), stilbenes – resveratrol (Fig. 6 A), isorhapontigenin, dimeric stilbene shegansu B,
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dihydroresveratrol. Simple phenols and phenolic glycosides have been also isolated: belallosides A and B:

acetovanillone 1-O- β-D-(6-O-vanilloyl) glucopyranoside and acetovanillone 1-O- β-D-(6-O-hydroxybenzoyl)

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glucopyranoside, respectively, and apocynin (Fig. 6 B), as well as a pyranocoumarin – decursin (Fig. 7B)

[15,16,21,31,34-38].

4.2. Triterpenoids

Iridal-type triterpenoids are characteristic constituents of Iridaceae plants including the genus Iris. There are

two forms of these compounds - free iridals and iridal esters. Free iridals are divided into four classes:

monocyclic iridals, cycloiridals, spiroiridals and oxaspiroiridals. Iridal-type triterpenoids have been also isolated

from Iris domestica [39-42]: iridotectoral B (Fig. 7A), iridotectoral A, iriditectoral, belamcandal, 28-

deacetylbelamcandal, 26-hydroxy-oxaspiroirid-16-enal, iridobelamal A, iridobelamal B, 26-hydroxy-15-

methylidene-spiroirid-16-enal, 26-hydroxy-13-oxaspiroirid-16-enal, 13-oxaspiroirid-16-enal, 16-O-acetyl-iso-

iridogermanal, iso-iridogermanal, 10-deoxyiridogermanal, 16-acetyl-3-decanoylisoiridogermanal, 16-acetyl-3-

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tetradecanoylisoiridogermanal, belachinal, anhydrobelachinal, epianhydrobelachinal, isoanhydrobelachinal,

spiroiridal, and a dimeric triterpenoid dibelamcandal A. Iridals were usually isolated from non-polar extracts or

fractions such as hexane, chloroform or diethyl ether using column chromatography on silica, Sephadex LH-20

and preparative HPLC on ODS phase.

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Beside Belamcandae chinensis rhizoma, other parts such as leaves and seeds of Iris domestica have been

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analyzed by only few research groups. The seeds contain p-benzoquinones (belamcandaquinones A and B) (Fig.

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8A), belamcandones A, B, C, and D (Fig. 8B) [15,43,44] benzofurans and their precursors –

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belamcandaphenols.

In the leaves, several isoflavonoids were identified: daidzin, genistin, sophoricoside, prunetin, tectoridin,

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genistein, iristectorigenin A, iridin, a few flavone glycosides – swertisin, 2”-O-rhamnosylswertisin, isovitexin,

2”-O-rhamnosylisovitexin, and flavonol glycoside – quercitrin. As in the rhizome, mangiferin was also present.
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[3,45,46].

4.3. Quantitative and qualitative analysis

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For isolation, structure elucidation and quantitative analysis of substances from different parts of Iris domestica,
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several traditional and advanced method were applied. Previously, most of the compounds were isolated using

classic preparative column chromatography on silica gel, Sephadex LH-20, ODS, with identification and
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structure elucidation using typical spectral methods (UV/VIS, IR, MS, NMR). For elution, several different

solvents were used, such as: hexane, chloroform, ethyl acetate, acetone, methanol. Subsequently, more advanced
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techniques have been also applied for isolation and purification of belamcanda constituents including

bidimensional preparative HPLC or HS-CCC (high-speed countercurrent chromatography) [33,47,48].

Nonetheless, it is still difficult to evaluate the advantage of these methods in terms of efficiency with respect to

the individual compounds.

When added all together [33,47,48], the amounts of pure polyphenols obtained from one kilogram of crude drug

have an unexpectedly wide range:

iridin –from 1.8 to 403 mg

tectoridin –from 4 to 2570 mg

tectorigenin –from 42 to 731 mg

irigenin –from 23 to 3200 mg

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irisflorentin –from 22 to 1560 mg

iristectorigenin B – ca. 375 mg

iristectorigenin A – 80-140 mg

mangiferin – ca. 900 mg

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The relatively large differences between the efficiency of preparative methods and the lack of verification of the

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actual recovery rates makes it impossible to suggest any optimal procedure in the current state of research.

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[28]. used a 2D-HPLC method for obtaining tectoridin, tectorigenin, iridin, irigenin, and irisflorentin. By using

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two or more columns switch, the authors claim to be able to faster collect pure compounds from crude extracts:

when two columns were used in the second dimension, in comparison to a single column in the second

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dimension or 1D-HPLC. However, the separation time was relatively long (over 100 minutes for each column),

especially compared to a regular preparative HPLC.

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In response to the demand for reliable data on the actual content of phytochemical markers in crude herb

samples, an LC-MS profiling has been performed by Li et al. [49]. In this paper, the authors provide qualitative
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information on 14 Belamcandae chinensis rhizoma samples collected in different regions of China. Twenty
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polyphenols were common for all samples and their HPLC fingerprints compared. These included sixteen

isoflavonoids, three xanthones and one stilbene – resveratrol. However, the authors’ assert that the percentage of
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the detected compounds is crucial for their impact on the herb quality remained in contradiction to the
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qualitative data, where some major bioactive isoflavonoids were not included. These omitted constituents are
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iridin, two glycosides iristectorin A and B, and one of the five most abundant aglycones – iristectorigenin A.

Instead, two xanthones – mangiferin and 7-O-methylmangiferin and resveratrol were included in the

quantitation. A reason for this selection was an uncertainty in identification of the remaining 13 compounds,

probably due to the lack of authentic standards. Unexpectedly, isorhapontigenin was not detected using the

reported methodology. These shortcomings would have to be worked out in future studies.

However, the complex chromatographic approach and using hyphenated techniques should be recommended for

phytochemical quality evaluation of drug samples, but also prior to preparative work for estimation of the extant

compound recovery. Authentication and elimination of adulterants such as other Iris sp. – for example I.

japonica [17] or I. tectorum (chuan she gan – used to treat sore throat and disperse phlegm) [50,51] is also an

important purpose of establishing a reliable and robust fingerprinting method. For these species, isoflavones

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with the 5,6,7 - trioxygenation pattern with variable O- substituents of 5,6,7 positions are more typical.

Differences are in their B-ring substitutions, especially with hydroxyl or methoxyl groups.

There are also substantial quantitative and qualitative differences resulting from harvesting season and region of

origin. According to the study by Qin et al. [52] isoflavone level reaches its maximum in spring rather than in

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fall. The herb from different provinces in China varied significantly [49]. Out of seven quantified compounds,

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the smallest range between highest and lowest content was for mangiferin (3.4-fold) and irisflorentin (5.9-fold).

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The highest exceeded one order of magnitude for such an important quality marker as tectorigenin (15.7-fold)

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and resveratrol (12.8-fold). Moreover, the proportions between some of the polyphenols were variable, for

example there was more tectoridin than irigenin in ten samples (tectoridin to irigenin ratio in mg/g, between 1.2

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to 15.6) and the opposite situation occurred in four (irigenin to tectoridin ratio from 1.2 to 2.0). This lack of

phytochemical uniformity is very likely to be reflected in the therapeutic value of the herbal drug, but the
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problem has not been thoroughly studied so far. Notwithstanding this diversity, in most of the cited

phytochemical research, iridin was the predominant compound.

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In the recent study by Chen et al. [53], the differential tissue distribution of several polyphenols was observed
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using laser microdissection of rhizome sections followed by LC-MS-based targeted metabolomic analysis.

Among the major compounds, mangiferin and a few hydrophobic isooflavone aglycons (irigenin,
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iristectorigenin A, tectorigenin) were located mainly in the cork, while isoflavone glycosides (iridin, iristectorin

B,tectoridin) were in the cortical layers, parenchyma, and around vascular bundles. Iridin and irigenin were also
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present in significant amounts in the roots. In the cork layer, the major peak in UHPLC-qTOF-MS was

irisflorentin, a compound which is otherwise not dominant in the entire rhizomes.

5. Biological activities of Belamcandae chinensis rhizoma.

5.1. Bioavailability

After oral administration tectoridin is hydrolyzed by intestinal microflora to tectorigenin that can be absorbed

into the blood and become an actual bioactive metabolite. In rats, tectorigenin was detected in the urine as a

major metabolite in amounts of about 1% of the orally administered tectoridin [54,55]. Biotransformation of

tectoridin to tectorigenin has been confirmed in vitro after incubation of tectoridin with intestinal flora and in

the liver microsomal incubation mixture. In vivo, after oral administration of tectoridin to healthy rats, six

metabolites of tectoridin in urine (tectorigenin, hydrogenated tectorigenin, mono-hydroxylated tectorigenin,

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di-hydroxylated tectorigenin, glucuronide-conjugated tectorigenin, sulfate-conjugated tectorigenin) and three

metabolites in faeces (tectorigenin, di-hydroxylated tectorigenin, sulfate-conjugated tectorigenin) have been

identified [56]. The highly methoxylated, lipophilic isoflavonoids are likely to be favored in absorption into a

mammalian organism, compared to non-methylated forms in a similar way to other flavonoids, for example a

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series of polymethoxyflavones from Citrus [57, 58], which also have superior anti-cancer and other

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pharmacological properties. However, the current literature does not provide sufficient data to support it. More

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bioavailability studies are necessary to know whether or not the belamcanda isoflavones are better absorbed

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than for example, the more common compounds from legumes.

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5.2. Antioxidative and antimutagenic.

So far, several papers have been published on antioxidant properties of either various extracts from
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Belamcandae rhizoma or isolated compounds thereof [20,21,59-66]. The main antioxidant compounds from

Belamcandae rhizoma belong to three polyphenol classes – isoflavonoids, xanthones, and stilbenes. The major
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phytochemicals in Belamcandae rhizoma – isoflavonoid glycosides and their aglycones, prevent excessive
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oxidation by various antioxidant mechanisms. They can efficiently reduce transition metal ions, decrease lipid

peroxidation, and/or scavenge free radicals. The fractions containing aglycons such as tectorigenin, irigenin and
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iristectorigenin were more active than glycoside fraction [64].

Among eleven popular TCM herbs, she-gan could be classified as a moderate scavenger of free radicals and
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active oxygen species, much weaker than the renowned TCM antioxidant drug – huang qin (Scutellariae

baicalensis radix) [66]. Nevertheless, the authors had screened only extracts enriched in glycosides (with

aglycones removed by prior chloroform washing) which are usually not as good antioxidants as the respective

aglycones.

In the ABTS scavenging assay coupled on-line with HPLC and MS/NMR compound identification, the most

active isoflavone was 5,6,7,3′-tetrahydroxy-4′-methoxyisoflavone, the remaining four antioxidant compounds

were tectorigenin, irigenin, tectoridin, and iristectorigenin [27,67]. Another screening with on-line analysis by

HPLC-DAD coupled with chemiluminescence and ESI-Q-TOF-MS/MS showed that the most effective

antioxidative compounds of Belamcandae rhizoma were iristectorin A, diglycosides of iristectorigenin A,

irigenin and irilin D [68]. However, two largest activity peaks (Iristectorigenin A 7-O-β-glucosyl(1→6)-

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glucoside and irilin D, respectively) corresponded to the minor compound peaks. Irigenin and iristectorigenin A

gave also significant antioxidant response but were among the major (first and fourth largest peaks) compounds.

This results evidently indicate that the contribution of an individual compound to the total antioxidative activity

is not directly related to its content.

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Tectorigenin, the most studied isoflavone from she-gan, prevents excessive oxidative processes by increasing

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antioxidant enzymes such as: SOD, CAT and GPx. Moreover, it is also a direct free radical scavenger.

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Tectorigenin is approximately two times more efficient DPPH scavenger than the glucoside – tectoridin.

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Furthermore, the radical scavenging activity of tectorigenin increases the survival ability of Chinese hamster

lung fibroblast (V79-4) cells under oxidative stress induced by hydrogen peroxide via activation of extracellular

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signal regulated kinase (ERK) pathway. Tectorigenin also increased the activities of antioxidant enzymes in the

V79-4 cells: superoxide dismutase, catalase and glutathione peroxidase by roughly 90%, 150% and 30%,
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respectively [62]. Moreover, tectorigenin was the only isoflavone (other tested compounds were irisflorentin,

irigenin, and tectoridin) able to protect a cultured hippocampal HT22 cell line from oxidative injury induced by
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the brain neurotransmitter glutamate. Tectorigenin showed a significant protective effect in a concentration-
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dependent manner, with an EC50 value of 67.25 µM [69].

Tectorigenin and tectoridin caused a significant decrease in serum transaminase activities (by about 40%) and
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lipid peroxidation (by 83% and 69% respectively) in CCl4 treated rats [59]. Both isoflavones induced also the
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activity of antioxidant enzymes (SOD, GSH-px, catalase) but had weak direct free radical scavenging potency
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expressed as IC50 values of 275 and 813 µM, respectively (compared to α-tocopherol IC50=10.4 μM).

The other major bioactive compound from Belamcandae rhizoma – mangiferin is typically obtained from

Mangifera indica – the mango tree and occurs in the popular tea from honeybush [70], but is also mentioned as

a bioactive marker of she-gan [1]. Most of the published studies used mangiferin from M. indica and showed

that it is both a direct and an indirect antioxidant [71].

The antioxidant properties of mangiferin are demonstrated by scavenging ROS, such as hydroxyl radical,

superoxide anion radical, or nitrosyl radical [60,61, 72-74], reducing transition metal (ferric) ions [75, 76] as

well as protection of organic substrates such as lipids and proteins from oxidative degradation [71].

The indirect counteracting of the Fe2+ - induced lipid peroxidation is based on strong chelating properties of

mangiferin, favored by a high ability to form a more stable Fe 3+-mangiferin complex and eventually inhibiting

the Fe3+ to Fe2+ reduction. Consequently, the level of the hydroxyl radicals generated by the Fenton reaction is

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significantly decreased (IC50 =9.02 µM). These in vitro studies suggest that the formation of the Fe3+ -

mangiferin complex unable to participate in the Fenton-type reaction contributes to the anti-peroxidative activity

of the mangiferin [77]. Additionally, under different experimental conditions, mangiferin- Fe3+ complex (2:1)

has been more effective than mangiferin itself in scavenging superoxide radicals generated by pyrogallol

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autooxidation and in protecting hepatocytes from reactive oxygen species- mediated hypoxia/reoxygenation

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injury (mangiferin- Fe3+ complex was 20% more effective than mangiferin alone) [78].

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Both stilbenes from Belamcandae chinensis rhizoma - resveratrol and isorhapontigenin are potent antioxidants.

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Isorhapontigenin can protect complex lipid molecules, such as LDL, or DNA from oxidative damage. The

protective effect was studied in hepatic microsomes, neuronal mitochondria and synaptosomes and was higher

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than exerted by a reference antioxidant – vitamin E [20]. It has also an inhibitory effect on the superoxide and

hydrogen peroxide production by activated rat neutrophils. The ability of this compound to directly scavenge
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oxygen free radicals (superoxide and hydroxyl) was confirmed by an electron spin resonance study [79].

Resveratrol has recently become the most studied stilbene and one of the most fashionable phytochemicals at
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all, with a vast existing literature, including clinical trials and numerous review papers [80-82]. However, she-
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gan is not a primary source of this compound, present mainly in such common products as grape skin and seed,

red wine, peanuts and another East Asian herb – Polygoni cuspidati rhizoma. However, among its numerous
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reported activities, antioxidative is one of the best documented in hundreds of papers [80, 83]. The reported
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mechanisms of the resveratrol antioxidant activity cover all known aspects, from metal chelation and reduction,
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scavenging of a variety of free radicals, preventing or chain breaking of lipid peroxidation to interaction with

antioxidant enzymatic mechanisms in the cells. Although resveratrol has been thoroughly studied as an isolated

compound from other plants, for the time being we cannot conclude its actual contribution to the overall activity

of she-gan. Therefore, there is a need for additional experimental studies that would elucidate the significance of

stilbenes for the total bioactivity of Belamcandae chinensis rhizoma.

Apocynin, a simple phenolic compound detected in Belamcandae chinensis rhizoma [1,84] is suggested to be a

selective inhibitor of NADPH oxidase (NOX). The enzyme plays an important role in the immune response by

reducing molecular oxygen to superoxide anion radical (•O2-) that is crucial for antimicrobial oxidative burst.

Therefore, apocynin could prevent excessive superoxide radical formation. NOX activity is the key free radical

generating process in the pathogenesis of vascular disorders (such as atherosclerosis). However, the actual mode

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of the apocynin action has been disputed, and its role as another direct antiradical compound is more likely in

this respect [85].

Mutagenic and antimutagenic properties of Belamcandae chinensis rhizoma were examined using several

bacterial assays both in crude solvent extracts and in isoflavone fractions and isolated compounds. Mutations in

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these assays can be induced directly by the test’s chemical – a direct mutagen or, in case of indirect

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mutagenesis, by metabolites of a promutagen after activation by hepatic microsomal enzymes (cytochromes

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P450). No genotoxic activity of the Belamcandae rhizoma has been detected using the Ames test with different

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Salmonella typhimurium strains or in Escherichia coli mutagenic assay. On the contrary, its antimutagenic

activity was observed against various chemical mutagens and UV radiation.

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Miyazawa et al. [86] have used the SOS umu test for bioactivity guided separation aiming at isolating minor

isoflavones with great antimutagenic properties against furylfuramide. As a result, using liquid-liquid extraction
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followed by the repeated column chromatography they have isolated iristectorigenin B as a primary antimutagen

in Belamcandae rhizoma methanol extract and further tested this compound and its methyl derivative using a set
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of bacterial assays. Both compounds have significantly, dose-dependently decreased the mutagenicity of the
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direct mutagen – furylfuramide, the enzymatically activated Trp-P-1, as well as of the UV irradiation.

Iristectorigenin B suppressed approximately 60% of the SOS-inducing activity caused by furylfuramide, 4NQO
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and MNNG, with ID50 values of 0.35, 0.21 and 0.23 µmol/ml, respectively. In the case of pro-mutagens, which
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require liver metabolic activation such as Trp-P-1 and AfB1 this isoflavone suppressed their mutagenicity by
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81% and 99% with the ID50 values of 0.42 and 0.26 µmol/ml, respectively. Interestingly, the same authors have

isolated tectorigenin as the main antimutagen from Pueraria lobata [87]. The contribution of tectorigenin – one

of the major isoflavones in she-gan in relation to the usually much less abundant iristectorigenin B, needs

clarification.

Both crude extract and isoflavonoid-enriched fractions obtained with selective SPE (solid phase extraction)

significantly inhibited direct and indirect mutagenesis in Ames tests, caused by NQNO and 2-AF, respectively

[63-65]. The predominance of glycosylated or aglyconic forms of flavonoids did not seem to be crucial in

determining the antimutagenic activity. The fractions containing more polar isoflavone glycosides were slightly

stronger than aglycone-rich fractions against the direct mutagen (up to 65% and 61%, respectively). All

fractions of the isoflavones in concentrations above 5 µg/mL, inhibited the indirect 2-AF induced mutagenesis

almost completely with no correlation to the glycosylation of isoflavones. Here, one of the likely mechanisms

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may involve the inhibition of the metabolizing enzymes rather than an interaction with the mutagen itself [88,

89].

Given the different mechanisms of the mutagenicity inhibition, the active constituents of Belamcandae rhizoma

can be classified as either bioantimutagens or desmutagens. The former term refers to the compound acting

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internally by protecting the genetic material from damage (e.g. caused by UV), while the latter rather prevents

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the mutagen attack or entry into the cell. The putative antioxidant mechanism involved in preventing the DNA

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oxidative injury by ROS or UV may be also considered as a contribution to the antimutagenic action.

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Other compounds contained in Belamcandae rhizoma, that are likely to contribute to the total antimutagenic

activity of its extract include resveratrol [88, 89] and mangiferin [65, 90]. Matsushima et al. [90] have reported

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mutagenic properties of xanthones from various medicinal plants. Only glycosylated xanthones – including

mangiferin were not mutagenic. Generally, in the presence of a hydrolytic enzyme (β-glucosidase), O-
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glycosides became mutagenic but mangiferin, a C-glucoside did not. The presence of the C-glycosidic bond

may be crucial for the compound safety. Moreover, mangiferin inhibited (by 56%) mutagenesis caused by the
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oxidative damage of DNA induced by H2O2 in TA102 strain, but not by NQNO. It can be explained by non-
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specific anti-ROS properties of mangiferin rather than by explicit antigenotoxic activity [65].

The summary of the Ames test results, showed that in most cases the tested isoflavones can be effective
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defensive against various mutagens, based on different mechanisms. Notwithstanding, solely iristectorigenin B,
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despite of the direct and indirect inhibition of the chemically induced mutagenicity, inhibits also the UV-
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induced mutagenesis and can be regarded as a bioantimutagen [86]. Taking together all results, the chemical

constituents of Belamcandae chinensis rhizoma act in concert to compose its significant antimutagenic

properties.

5.3. Phytoestrogenic

Experimental data confirm the estrogenic properties of Belamcandae rhizoma, especially of isoflavonoid

aglycones – irigenin and tectorigenin. Among 32 TCM herbs tested with transgenic yeast system expressing

estrogen response plasmid, the alcoholic extract from Belamcandae rhizoma was the most potent. Although in

terms of the EC50 value and relative potency it was ranked behind seven other herbs, it had the highest

estrogenic inductive efficiency expressed as the highest reporter gene response in comparison to β17-estradiol

(83.7% of estradiol activity) [91].

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The chemical structure, particularly the distribution and number of hydroxyl groups of molecules similar to

17β–estradiol, affects the estrogenic activity. The role of hydroxylation patterning in isoflavones (tectoridin,

tectorigenin, irilin D, iridin irigenin, irisflorentin, iristectoridin A) and a stilbene (resveratrol) from she-gan in

estrogenic properties in cells has been recognized by 2D- and 3D-QSAR analysis [92]. The hydroxylation of 3’

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and 5’ carbons of the B ring and the inter-atomic distance between the hydroxyl groups attached to the outer

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terminal position 7 of the A ring and 4’ of the B ring of the isoflavonoid scaffold is essential for the estrogenic

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activity. In this study, tectoridin among the set of 55 estrogen-like compounds obtained from Thai medicinal

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plants was the second (after genistein) most potent phytoestrogen in MCF-7 breast cancer cell line proliferation

assay with affinity towards both ER α and ER β receptors. Tectorigenin was also one of the most active tested

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isoflavones whereas the other such as iridin, irilin D were moderate, and irisflorentin, irigenin and iristectoridin

B were considerably weaker. The strongest phytoestrogens – tectoridin and tectorigenin, have hydroxyl group in
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4’ position but only the aglycone has free hydroxyl group in C-7 position. Both isoflavones with free OH groups

in C7 and C3’ (irilin D, irigenin) were weaker. Tectoridin (no free 7-OH group) had only negligible affinity to
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both ERα and ERβ. Therefore, the mechanism of the higher estrogenic activity of a glycosylated form
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(tectoridin without a free 7-hydroxyl group) over tectorigenin in cell culture has not been explained in this

study. Compared to genistein - a reference phytoestrogenic isoflavone, belamcanda isoflavones have lower in
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vitro binding efficiency towards ERα and ERβ. However, tectoridin, tectorigenin, iridin and irigenin despite
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their lower affinity to estrogen receptors α and β exert a strong estrogenic effect [93, 94].
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A number of earlier studies [93, 95, 96] have demonstrated that the estrogenic activity of pure tectorigenin from

Belamcandae rhizoma does result from a strong binding to ERs α and β. Belamcandae rhizoma and its

constituents can inhibit prostate cancer cells proliferation as well as put under control the deregulated expression

of some genes related to neoplastic transformation of prostate cells. In the study by Morrissey et al. [97]

tectorigenin and irigenin caused G1 arrest and induction of proapoptotic proteins in three prostate cancer cell

lines. The mechanism of this activity via estrogen receptors could not be confirmed, though.

In animal in vivo studies, the mice were injected subcutaneously with LNCaP cells. Simultaneously, the mice

were fed with B. chinensis extract (containing 5% of tectorigenin), and the average daily intake was 37 mg per

animal [96]. After 6 weeks, the inhibition of transplanted tumor growth was observed. Some of the treated

mice had remained tumor free for the whole duration of the experiment. The tumor inhibitory effect was

concurrent with a 35% reduction of PSA levels in the serum, but of no statistical significance.

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These in vivo results are supported by the data from in vitro human and murine tumor cell lines studies, where

the tested belamcanda isoflavoids rectified the aberrant expression of several genes involved in the proliferation

of cells and the development of prostate cancer. In LNCaP (androgen sensitive human prostate carcinoma cells)

cell cultures, the Belamcandae rhizoma extract and several belamcanda isoflavonoids, down regulated the

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androgen receptor expression on both mRNA and protein level, the PSA expression and secretion, as well as the

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expression of the prostate cancer specific marker DD3PCA3 and PDEF (prostate derived Ets factor) which is a

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transcription factor associated with oncogenesis in prostate cells [96, 98-100]. In addition, the IGF-1 receptor

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(insulin-like growth factor 1 receptor) mRNA expression and androgen-dependent hTERT (a telomerase

subunit) mRNA expression were strongly reduced by treatment with belamcanda phytochemicals. The authors

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have also observed a decreased expression of NKX3-1, another transcription factor and putative prostate tumor

suppressor, whose role in the overall anticancer effect of Belamcandae rhizoma has not been elucidated, though.
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Additionally, the isoflavone aglycones (tectorigenin, irigenin, and iristectorigenin) from markedly increased the

in vitro expression of a pro-apoptotic metalloproteinase factor TIMP-3 encoding gene in LNCaP cells. These
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effects on the gene expression level were concomitant with a decreased proliferation of LNCaP cells [94].
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Therefore, both the extract and a few purified isoflavones exert these beneficial effects on the expression of

genes relevant in proliferation and oncogenesis.

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In animal in vivo studies, the mice were injected subcutaneously with LNCaP cells. Simultaneously, the mice
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were fed with B. chinensis extract (containing 5% of tectorigenin), and the average daily intake was 37 mg per
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animal [96]. After 6 weeks, the inhibition of transplanted tumor growth was observed. Some of the treated

mice had remained tumor free for the whole duration of the experiment. The tumor inhibitory effect was

concurrent with a 35% reduction of PSA levels in the serum, but of no statistical significance.

The apparent discrepancy between the relatively low affinity of the belamcanda isoflavones to ER, and a strong

phytoestrogenic activity draws our attention to the complex mechanisms of their physiological effects which are

still not fully understood. Most of the cited studies, report estrogenic activities based on ER-signaling. However,

the ER-binding efficiency is not always in direct relation to the resulting physiological outcomes. For example,

tectorigenin and formononetin, have a similar ERα and β binding strength, whereas the estrogenic effects

exerted through this ER pathway are stronger in the latter compound [92, 93]. Tectoridin hardly binds to ERα

compared to 17β-estradiol and genistein [93]. Conversely, genistein and estradiol stimulate the genomic

estrogen signaling pathway via ER receptors to a much larger extent than tectorigenin because of a high binding

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efficiency towards ERα and ERβ. Despite having weak binding affinities to ER, both tectorigenin and tectoridin

exerted meaningful estrogenic effects [11,95].

The estrogen receptor pathway is not the only way of phytoestrogen activity in the organism. In the ER-

pathway, the estrogen (or phytoestrogen) binds to the receptors, alters their conformation, and the resulting

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complex interacts with estrogen-responsive elements in the DNA causing modification in the gene expression

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patterns followed by certain physiological events. This is the genomic estrogen signaling pathway. In contrast,

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the non-genomic signaling is based on binding of an estrogen to a transmembrane G-protein coupled receptor

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named GPR30, causing fast intercellular signals that activate several targets such as EGFR (epidermal growth

factor receptor), kinases (ERK) and NfκB (nuclear factor kappa B). This rapid non-genomic signaling is also

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widely observed following stimulation of cells and tissues with estrogen and the GPR30 has become recognized

as an estrogen receptor complementary to the classical estrogen receptor (ER) [101]. Both genomic and non-
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genomic pathways are interconnected and act in concert to bring about the complexity of the final estrogenic

response. It may explain the estrogen effects of tectoridin and tectorigenin probably transmitted in the greatest
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extend via the GPR30 way [94].

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Tectorigenin, affecting the hypophysis activity influences such estrogenic responses as the inhibition of pulsate

LH secretion correlated with hot flushes and calcium absorption in bones, especially in menopausal women.
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These beneficial properties are not accompanied by induction of undesired alterations in the uterus and the
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mammary glands [95]. Hence, it could be concluded that tectorigenin a weak ER ligand, but a potent estrogenic
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effects stimulator, could become useful as an alternative category of selective estrogen receptor modulators

(SERM). On the other hand, the GPR30 pathway apart from being involved in regular physiological estrogen

response, is highly activated in carcinogenesis progression, especially in breast and endometrium cancers, that

are potential targets for the pathway modulators of phytoestrogenic origin [101, 102]. Hence, using this

compound as SERM should be considered with some precaution.

In addition to isoflavonoids, decursin - a pyranocoumarin contained in small amount in Belamcandae chinensis

rhizoma was studied for its chemopreventive potential in hormone-dependent breast and prostate cancers.

Decursin inhibited estrogen-stimulated carcinogenesis on independent MDA MB-231cells. This compound

arrested the cell cycle as well as induced apoptosis through an increased expression of ERβ. The anticancer

efficacy of decursin via the cell cycle arrest and apoptosis induction has been also shown in human prostate

carcinoma DU 145, PC-3 and LNCaP cells [103, 104].

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The phytoestrogenic activity of Belamcandae rhizoma is high and has a great potential for being introduced into

the official evidence-based complementary medicine. In most of the published data, it has been emphasized that

Belamcandae rhizoma and its isoflavones have no side effects and are generally considered as safe [7]. At the

same time, our knowledge about the physiological, biochemical, and genetic mechanisms of phytoestrogens

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role, both in healthy and pathologically affected organisms, is still incomplete. In most cases, we know the final

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effect without realizing the specific mechanisms underneath. The aforementioned current state of research

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provides a substantial base for continuing studies on the cellular and in vivo mechanisms of action as well as on

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preventive and therapeutic potential of Belamcandae rhizoma phytoestrogens.

5.4. Other activities

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5.4.1. Anti-inflammatory

The traditional indications of Belamcandae chinensis rhizoma suggest that antiinflammatory mechanism of
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action might be involved in its therapeutic properties. The major isoflavone aglycones - irigenin and

tectorigenin, and the glucoside tectoridin were studied for their anti-inflammatory properties [105-108]. In the
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presence of tectorigenin or tectoridin, the prostaglandin E2 production, stimulated by TPA (12-O-

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tetradecanoylphorbol 13-acetate) or thapsigargin, decreased in a concentration-dependent manner (3, 10 and

30µM). Tectoridin and tectorigenin did not affect the release of arachidonic acid from the membrane
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phospholipids of macrophages and showed no direct inhibitory effect on COX-1 and COX-2 activity. Both
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compounds inhibited the induction of COX-2 protein (but not COX-1) [105]. Therefore, tectoridin and
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tectorigenin act by influencing the inflammation mediators. The COX-2 dependent PGE2 production by

macrophages is selectively inhibited, but the mechanism involves the inhibition of NF-κB and respective gene

expression, rather than a direct COX-2 inhibition. This type of activity is likely to induce a broad anti-

inflammatory response by decreasing both the level of PGE2 and other molecules, such as pro-inflammatory

cytokines and NO. Indeed, in the Raw 264.7 cells the production of PGE2, NO, IL-1β induced by IFN-γ/LPS

was decreased in the presence of 3-30 µM of irigenin and 50-200 µM of tectorigenin. These effects were

caused by lowering the expression level of iNOS and COX-2 mRNA and proteins rather than their enzymatic

activity. In contrast, tectoridin and genistein showed no inhibitory effect in almost all anti-inflammatory tests

[109, 110]. Irisflorentin, an isoflavone from Belamcandae rhizoma hardly mentioned in scientific literature,

significantly decreased the pro-inflammatory cytokine production such as tumor necrosis factor α , interleukin-

6, and interleukin-12p70 stimulated by LPS in dendritic cells (DCs). The findings also showed that irisflorentin

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can modulate an aberrant activation of DCs and through this capability prevent a harmful immune response. The

treatment with up to 40 µM of irisflorentin does not cause cytotoxicity [111].

Mangiferin is also an inhibitor of inflammatory processes involving prostanoid signaling mediated by pro-

inflammatory cytokines such as TNF-α and NFкB activation [112-114]. In fact, as in the antioxidant activity,

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the majority of the published work has regarded the mango tree as the only source of mangiferin. Its presence in

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considerable amounts also in Belamcandeae rhizoma, suggests that this compound may contribute significantly

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to the final, multifaceted activity of the drug. However, this assumption requires further studies to conclude.

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5.4.2. Anti-angiogenic, cytotoxic, and adhesion-inducing

5.4.2.1. Isoflavones

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In a single study by Jung et al. [115], tectorigenin and tectoridin were compared to genistein for the inhibition of

various aspects of angiogenesis, including de novo vascularization of solid tumors in mice and their anti-
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proliferative effect. Tectorigenin was stronger than tectoridin as an angiogenesis inhibitor in both mouse and

egg models, as well as reduced the size of tumors, albeit to a lesser degree than reference drugs (5-fluorouracil
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and doxorubicin). Tectorigenin considerably and dose dependently, inhibited calf pulmonary arterial
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endothelial (CPAE) cell growth with the IC50 value 67.9 µM (compared to genistein, IC50 value 66.9 µM).

Furthermore, tectorigenin, injected subcutaneously at the dose 30 mg/kg for 20 days to mice implanted with
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murine Lewis lung carcinoma (LLC), caused a significant (30.8%) inhibition of tumor volume. Both
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tectorigenin and tectoridin cause in ICR mice bearing sarcoma 180, a suppression in tumor weight by 44.2 and
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24.8%, respectively. The putative mechanism of this kind of activity may base on the inhibition of endothelial

cell proliferation, which is an important step in angiogenesis. This mechanism was also related to the COX-2

inhibitory properties of studied isoflavones [115].

5.4.2.2. Triterpenoids

Ito et al [42] studied the ichthyotoxic properties of iridals and reported the fish killing ability of belachinal, (+)-

(6R,10S,11S,14S,26R)-26-hydroxy-15-methylidenespiroirid-16-enal and 16-O-acetylisoiridogermanal.

Although most of monocyclic iridals appeared to be nontoxic to fish the 16-O-acetylisoiridogermanal turned out

to be the one toxic. The mentioned above compounds were highly toxic to killie-fish (Oryzias latipes). The

dimeric iridal triterpenoid - dibelamcandal A is also toxic to the freshwater snail Pomacea canaliculata [116].

Three of the iridals (of 6 tested) exhibited a significant ability to induce cell adhesion in HL-60 cell cultures

[39]. 28-deacetylbelamcandal, 26-hydroxy-15-methylidene-spiroirid-16-enal and 26-hydroxy-13-oxaspiroirid-

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16-enal were active and induced 100% HL-60 cell-adhesion in concentration of 0.5, 1.3 and 2.5 µM

respectively. In another study by Takahashi et al. (2002) the activation of protein kinase C (PKC) was observed

for a few tested iridal type triterpenoids. Among four classes of iridals, spiroiridals exhibited the highest

activation of PKC and the 28-deacetylbelamcandal (ED50=300 nM) was the most effective compound, followed

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by 26-hydroxy-15-methylidene-spiroirid-16-enal and 16-O-acetyl-isoiridogermanal. The authors found the 28-

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deacetylbelamcandal to be a new class TPA-type tumor promoter as well as a PKC activator referring to TPA

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(ED50=20nM) activity. This type of action is considered unfavorable when it comes to its role in tumor

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stimulation and growth promotion. The results even suggested that spiroiridals are candidates for chemical

tumor promoters. However, the content of their in the Belamcandae rhizoma is low (7 miligrams of 28-

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deacetylbelamcandal was obtained from 1 kg of rhizomes) and being among the most hydrophobic, the

potentially harmful iridals are contained in non-polar extracts. The hydrophobic side chain is suggested to be
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crucial for PKC binding activity [117]. However, for safety reasons, the herbal formulae should be routinely

tested for the mentioned iridals.

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5.4.3. Activities related to the respiratory tract

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Despite the primary indication in the TCM, there are only few reliable reports confirming this type of activity in

vitro and in vivo in animal studies.

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In the study by Surender and Agrawal [118], the extracts of Belamcanda chinensis (B.C.) were evaluated for
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bronchorelaxant property against histamine aerosol induced bronchospasm. The aqueous and 50% ethanolic
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extracts of its leaves and rhizomes exhibited a significant protection against histamine induced pre-convulsive

dyspnoea in guinea pigs. In a model of asthma in guinea-pigs a complex decoction containing Belamcandae

chinensis rhizoma as one of the constituents (Wuwei Dilong) inhibited infiltration and diffusion of the

inflammatory cells and regulated LTB4 and IFN-γ levels [49].

Tectorigenin inhibits the proliferation of pulmonary fibroblasts from bleomycin-treated rats in vitro and

enhances the expression of miR-338* (a type of microRNA hypothetically involved in regulation of pulmonary

fibrosis progression), which might in turn post-transcriptionally downregulate LPA1 – a protein that plays a

critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). This indicates a potential inhibitory role

of tectorigenin on the pathogenesis of IPF [10].

5.4.4. Antibacterial and antiviral.

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Tectorigenin inhibits the growth of dermatophytes such as Trichophyton sp. Saccharomyces sp. and Candida

sp. [119]. The minimum inhibitory concentration (MIC) of tectorigenin was in the range of 3.12-6.25 µg/ml

against Trichophyton sp. and 25-50 µg/ml against such yeast-like fungi as Candida sp. and Saccharomyces sp.

An insignificant antifungal activity of this isoflavone was noted against Aspergillus niger (MIC>200 µg/ml).

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The moderate antibacterial activity of tectorigenin was also revealed against Pseudomonas aeruginosa, Proteus

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vulgaris, Micrococcus luteus, and Staphylococcus aureus (50-100µg/ml). Compared to some other flavonoids,

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such as kaempferol, quercetin or naringenin, the antimicrobial activity spectrum of tectorigenin is not inferior

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but broader [119]. Moreover, tectorigenin showed antibacterial activity against methicillin-resistant

Staphylococcus aureus (MRSA) but the MIC was quite high - 125 µg/ml. Combining tectorigenin (6.25 µg/ml)

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and detergent TX-100 highly enhanced the antibacterial activity against MRSA . The anti-MRSA action of

tectorigenin was suggested to result from increase of the cytoplasmic membrane permeability and inhibition of
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ATPase [120]. These results revealed tectorigenin as a promising compound for prospective in vivo experiments

and a potential clinical treatment of tectorigenin on MRSA infected patients.

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According to [74], mangiferin has also some antimicrobial properties against bacteria: Staphylococcus aureus,
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Staphylococcus citreus, Escherichia coli, Salmonella agona, Klepsiella pneumoniae and fungi: Saccharomyces

cerevisiae, Thermoascus aurantiacus, Trichoderma reesei, Aspergillus flavus, and Aspergillus fumigatus.
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Mangiferin was also active in vitro against viruses such as HSV, HIV and influenza [48,121].
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5.4.5. Hypoglycemic
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A few reports also suggest an antidiabetic potential of Iris domestica, but besides rhizomes, the leaves were

considered as source of the same bioactive compounds. The aqueous extract from leaves of Iris domestica exert

the hypoglycemic action in both healthy and STZ-induced diabetic rats [3,45]. The extract enhanced the

secretion of insulin significantly in a dose dependent manner. This stimulation of insulin secretion is the most

likely mechanism for lowering of blood glucose. The extract decreased blood glucose levels by 30% in the dose

range of 0.4-1.6 g/kg. The most effective dose was 1.6 g/kg but a dose above 3.2 g/kg became ineffective. The

efficiency of the extract was even higher when administrated via intraperitoneal injection. One i.p. treatment

with the extract (125 and 250 mg/kg) gave a reducing blood glucose effect equivalent to a multiple oral

administration of 1.6 g/kg. The bioactivity guided fractionation allowed to attribute the activity to isoflavonoid

glycoside - tectoridin and flavone glycoside – swertisin, as well as to the xanthone – mangiferin. The IC50 values

show that hyperglycemia inhibitory effect of isoflavones (250 mg/kg) was comparable to positive control - 100

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mg/kg of Acarbose [46]. Tectoridin and tectorigenin inhibited aldose reductase – the key enzyme in the

pathogenesis of diabetic complication with IC50 values 1.08 μM and 1.12 μM, respectively. Other flavonoids

isolated from Belamcandae chinensis rhizoma in the same study were significantly less active [120].

The antidiabetic properties of mangiferin such as reducing the glucose concentration in plasma and

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improvement of oral glucose tolerance without inducing hypoglycemia were also reported in rats [123,124]. The

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hypoglycemic activity may result from both pancreatic and extrapancreatic mechanisms, additionally enhanced

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by high antioxidant capacity [71]. The actual role of these interesting compounds in the antidiabetic properties

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of Belamcandae chinensis rhizoma or leaves of the same plant still requires elucidation.

5.4.6. Allelopathic

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Somehow aside of the pharmacological properties is the allelochemical activity of belamcanda. Kim et al. [125]

reported the enhancement of heavy metal uptake by a weed-grass Echinochloa crus-galli by belamcanda root
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exudates. The water extract from belamcanda roots soaked for 48 hours improved phytoextraction of soil

contaminated with Pb, Cd and Cu. The authors attribute this activity to the presence of carboxylic acids such as
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oxalic, citric, succinic and acetic, and their chelating properties. They have not, however, tested the extract for
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water soluble polyphenols, such as isoflavone, xanthone or stilbene glycosides, that could also contribute to the

reported phenomenon. If indeed any of the polyphenol glycosides was present in the exudates fraction, it would
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be another interesting role of such compound both in the plant itself and in the environment.
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6. Conclusions
AC

In conclusion, the knowledge about Belamcandae rhizoma has been growing rapidly over the recent few years,

but there are still significant gaps in the completeness of our understanding of its bioactivity, therapeutic value,

and roles played by each of the numerous polyphenols. Most of the studies are fragmentary, or focused only on

few isolated compounds. It is still unclear which of the compounds determined its traditional therapeutic

applications in East Asian phytotherapy systems. These “traditional medicines” have recently gained popularity

in different parts of the world facing problems with lifestyle – related diseases and limited resources for equal

access to health care. There is a demand for high-quality herbal drugs derived from East Asian plants.

Belamcandae rhizoma is one of the important herbal medicine in East Asian phytotherapy systems, such as

TCM, Kampo, Korean, Thai, Nepalese and Vietnamese. Information on its therapeutic properties and use, based

on the original descriptions does not fit into the requirements of contemporary, evidence-based medicine. Even

so, it supports the potential of this herb resulting from extensive experience and apparent lack of side effects. In

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recent times, due to its worldwide use, potential impact on healthcare and opportunities for new drug

development, Belamcandae chinensis rhizoma has already been included in the European Pharmacopoeia

monographs.

Highly methoxylated isoflavonoids, xanthone glucosides, and stilbenes should be listed as major polyphenolics,

T
that influence its pharmacological profile. The most abundant among them are numerous isoflavone glycosides

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and aglycones, unique in their diverse hydroxylation and methoxylation patterns of A and B-rings of isoflavone

R
skeleton. Generally, isoflavonoids are considered to be major constituents of Belamcandae chinensis rhizoma

SC
responsible for its main estrogenic and anti-inflammatory effects and are regarded as indicators for quality

assessment of this drug.

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The predominance of highly methoxylated forms makes this herb a good candidate for alternative therapy due to

their supposedly better bioavailability.

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In our opinion, mangiferin as one of major components should be also taken into account in characterization of

the overall properties of this herb. Its valuable activities confirmed by numerous studies, with antiinflammatory
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action in the first place, can explain at least some of the traditional uses.
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The presence of a number of iridal-type triterpenoids in Belamcandae rhizoma should be considered in order to
P

verify their participation in the bioactive profile of the drug. There is a necessity of a comprehensive
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characteristic of pharmacological properties of these compounds. To the best of our knowledge, only a few
AC

biological activities such as cytotoxicity, ichthyotoxicity and PKC activation exhibited by iridals have been

reported in Belamcanda chinensis. Of particular interest remain the unfavorable growth promotion and tumor

stimulation properties of the spiroiridals.

Given the long ethnobotanical history of use of this herb in a variety of diseases, the recent interest in this plant

as an object of pharmacological and phytochemical studies is fully justified. The most significant and well

described property of these compounds is the modulation of estrogen response. The isoflavones irigenin,

tectorigenin (and glucosides of them) could find their place as phytoestrogens, alternative to soybean or clover.

For this, the more detailed physiological and pharmacological studies are needed. We have to know exactly

which signaling pathways they affect, and what role they play in pathogenesis of hormone-dependent disorders.

Additionally, the antimutagenic and antioxidant activities suggested a preventive value of she gan in protection

from early genotoxic events and oxidative stress.

23
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As above described in detail, our knowledge of molecular and cellular targets of active principle from she gan is

at the moment limited to the involvement of isoflavones in estrogenic and mangiferin in antiinflammatory

signaling. Hence, more discoveries are expected in the near future that would elucidate the mising … …. …

The relatively well described phytoestrogenic activity is in striking discrepancy to the principal indication in

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TCM which is the respiratory tract. Despite it, the research published in the international literature supporting its

IP
use in such diseases as upper respiratory tract or pulmonary infections, sore throat and asthma, is scarce. Only

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few reports on its anti-inflammatory activity could be in favor of this traditional usage. The herb, used in several

SC
regions of East Asia for centuries, has remained almost unheard of in other parts of the world until very

recently.

NU
In our opinion, further research should focus on the elucidation of the complex relations between various

activities and the phytochemical composition. Not only the isolation and structure elucidation of individual
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compounds, followed by pharmacological screening, but also the deep understanding of interactions, synergies

and antagonisms of the entire complex matrix should attract the researchers’ interest. In addition, the
D

contribution of B. chinesis rhizoma and its individual compounds to the therapeutic value of composite
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preparations such as TCM patent drugs, frequently used in traditional herbalism should be explored. A possibly

complete understanding of correlations between pharmacological properties and phytochemical profile would
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enable reasonable application of this herb in modern phytotherapy.

CE

Acknowledgements
AC

Funding: This publication is based on work supported by Wroclaw Medical University grants ST-695, ST-696

and ST-909.

Compliance with Ethical Standards

Conflict of Interest: The authors declare that they have no conflict of interest.

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Captions to figures

Fig.1 Structures of six major isoflavones identified in the Belamcandae chinensis rhizoma

Fig.2 Structures of all isoflavones found in Belamcandae chinensis rhizoma. Me=CH3, Glc=glucose

No compound R1 R2 R3 R4 R5 R6 R7
1 tectorigenin H OMe OH H H OH H

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2 8-hydroxytectorigenin H OMe OH OH H OH H

3 tectorigrnin-7-O-glucosyl-4-O-glucoside H OMe OGlc H H OGlc H

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4 tectorigenin-4’-O-glucosyl(1→6)glucoside H OMe OH H HO OGlc-Glc H

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5 tectorigenin-7-O-βglucosyl(1-6)glucoside H OMe OGlc-Glc H H OH H

6 tectorigenin-4’-O-β-glucoside H OMe OH H H OGlc H

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7 4’,7-di-O-methyltectorigenin H OMe OMe H H OMe H

8 isotectorigenin H H OH OMe H OH H
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9 isotectorigenin-7-O-β-D-glucoside H H OGlc OMe H OH H

10 3-‘hydroxytectoridin H OMe OGlc H OH OH H

11 tectoridin H OMe O-Glc H H OH H

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12 homotectoridin H H OGlc OMe OMe OH H

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13 psi-tectorigenin H H OH OMe H OH H

14 irigenin H OMe OH H OH OMe OMe

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15 8-hydroxyirigenin H OMe OH OH OH OMe OMe

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16 isoirigenin H H OH OMe OH OMe OMe

17 iridin H OMe O-Glc H OH OMe OMe

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18 isoiridin H H O-Glc OMe OH OMe OMe

19 iristectorin A H OMe O-Glc H OH OMe H

20 iristectorin B H OMe O-Glc H OMe OH H

21 iristectorin B-4’-O-glucoside H OMe OGlc H OMe OGlc H

22 iristectorigenin A H OMe OH H OH OMe H

23 isotectorigenin B H OMe OH H H OMe OH

24 dalspinosin C H OMe OH H H OMe OMe

25 8-hydroxyiristectrigenin A H OMe OH OH OMe OH H

26 iristectorigenin A-7-O-glucoside H OMe OGlc H OMe OH H

27 iristectorigenin A-7-O-glucosyl(1→6)glucoside H OMe OGlc-Glc H OH OMe H

28 iristectorigenin B H OMe OH H OMe OH H

29 iristectorigenin B-7-O-β-glucosyl(1→6)glucoside H OMe OGlc-Glc H OMe OH H

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30 iristectorigeninB-4’O-β-glucosyl(1→6)glucoside H OMe OH H OMe OGlc-Glc H

31 belamcandinin H OMe OMe H OMe OMe H

32 5,7,3'-trihydroksy-8,4'-dimetoxyisoflavone H H OH OMe OH OMe H

33 irisolidon H OMe OH H H OMe H

34 methylirisolidon H OMe OMe H H OMe H

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35 2,3-dihydroirigenin H OMe OH H OH OMe OMe

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36 irilin D H OMe OH H OH OH H

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37 5,7,4’-trihydroxy-6,3’,5’-trimethoxyisoflavone H OMe OH H OMe OH OMe

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38 5,6,7,3' tetrahydroxy-4'metoxyizoflavone H OH OH H OH OMe H

39 5,7,8,4’-tetrahydroxy -6-methoxyisoflavone H OMe OH OH H OH H

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40 junipegenin C H
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Fig. 3 Structures of isoflavones from Belamcandae chinensis rhizoma with additional dioxolane ring.

No. compound R1 R2 R3 R4
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41 irisflorentin OMe OMe OMe OMe

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42 noririsflorentin OH OMe OMe OMe

43 dichotomitin OH OH OMe OMe
44 irilone OH H OH H
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45 3’,5’-dimethoxyirisolone 4’-O-β-glucoside OH OMe OGlc OMe

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Fig. 4 Structures of acylated iridin glycosides: A- 6”-O-vanilloyliridin, B- 6”-O-p-hydroxybenzoyliridin

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Fig. 5 Structures examples of flavones and flavonols.

No. compound R R1 R2 R3 R4 R5
1 luteolin H H H OH OH H
2 apigenin H H H OH H H
3 hispidulin H OH OMe OH H H
4 5,4’-dihydroxy-6,7-methylenedioxy-3’-methoxyflavone H OH O-CH2-O H OMe
5 5,7,4’-trihydroxyflavanone H H H OH H H
6 isorhamnetin OH OH H OH H OMe
7 rhamnazin OH H OMe OH OMe H
8 rhamnocitrin OH OH H OMe H H
9 astragalin OGlc OH H OH H OMe
10 kanzakiflavone-2 H OH O-CH2-O H H

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Fig. 6 Structures of various polyphenols from Belamcandae rhizoma. A- trans-resveratrol (stilbene), B-
acetovanillone – apocynin, and xanthones: C – isomangiferin, D – mangiferin, E - neomaniferin

Fig. 7 Structures of : A - iridotectoral B, an example of iridal-type triterpenoid, and B - decursin

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Fig. 8 Structures of quinone compounds from seeds of Iris domestica.

A. Belamcandaquinones : R= -(CH2)9CH=CH(CH2)3CH3. Belamcandaquinone A: R1= OMe, R2= R.

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Belamcandaquinone B: R1= CH2)9CH=CH(CH2)3CH3, R2= OMe

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B. Belamcandones:

Belamcandone A: R1= R2= -(CH2)9CH=CH(CH2)3Me, R3= H

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Belamcandone B: R1= -(CH2)9CH=CH(CH2)3Me, R2= -(CH2)11CH=CH(CH2)3Me, R3= H

Belamcandone C: R1= -(CH2)9CH=CH(CH2)3Me, R2= -(CH2)16Me, R3= H

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Belamcandone D: R1= -(CH2)11CH=CH(CH2)3Me, R2= -(CH2)16Me, R3= H
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