2008, Vol.13 No.

2, 239-246

Article ID 1007-1202(2008)02-0239-08
DOI 10.1007/s11859-008-0221-2

Identification of Mucilage Cavity

as a Significant Microscopic
Characteristic Existing in Phloem
Instead of Pith of Radix et Rhizoma Rhei

ƶ TANG Jun1,2, FENG Yibin1, TONG Yao1, 0 Introduction

JIA Ruirui1, SY Laiking3, R. Y. K. Man3†
1. School of Chinese Medicine, The University of Hong Kong, Hong
Kong, China;
2. College of Pharmacy, Wuhan University, Wuhan 430072, Hubei,
China;
3. Department of Pharmacology, The University of Hong Kong, Hong
Kong, China
Abstract: The morphologic and microscopic features of Radix et Rhizoma
Rhei were studied. The study verified that the mucilage cavities did exist in
phloem of rhizomes and roots or abnormal vascular bundles. Also, they were
in similar distribution in three species of Radix et Rhizoma Rhei. The diagnos-
tic characteristic for microscopic identification was found to be the similar
distribution of abnormal vascular bundles in pith of rhizomes in all three spe-
cies. And the appearance of the crude drug varied more depending on the
plants’ geographical origin and different preliminary treatment on the spot of
collection than on the species differences. Our findings, having not been de-
lineated clearly so far in the previous reports, are helpful for clarifying current
descriptions in different literatures or standards and make a full understanding
on microscopic and macroscopic identification of Radix et Rhizoma Rhei.
Key words: Radix et Rhizoma Rhei; microscopic identification; mucilage
cavity; abnormal vascular bundle
CLC number: R 93; R 9315
Received date: 2007-04-25
Foundation item: Supported by the Hong Kong Chinese Materia Medica Standards
Project of Department of Health of Hong Kong Special Administrative Region Government
(00600005) and Seed Funding of School of Chinese Medicine, the University of Hong Kong
(92210. 22100. 01).
Biography: TANG Jun (1970-), male, Associate professor, Ph.D., research direction: phar-
macognosy, Chinese medicine and chemical pharmacology. E-mail: tangj0205@hotmail. com
† To whom correspondence should be addressed. E-mail: rykman@ hkucc.hku. hk
Radix et Rhizoma Rhei, known as ‘Da-
huang’ in Chinese or rhubarb in English, is one of
the most important medicinal herbs in the world.
The high-quality crude drugs are indigenous in
China. Usually, the officially registered medicinal
herb is dried rhizome and root of three species of
section Palmata A. Los., belonging to Rheum
(Polygonaceae), which is, Rheum palmatum L.
(‘Zhangye Dahuang’), Rheum tanguticum Maxim.
ex Balf. (‘Tanggute Dahuang’) and Rheum offi-
cinale Baill. (‘Yaoyong Dahuang’) and can be
used to treat fever with constipation, dysentery
and jaundice[1-5].
For the purpose of authentication of the
crude drugs, the macroscopic, organoleptic and
especially microscopic identification have been
or still being adopted in the Chinese Pharmaco-
poeia and many other international Pharmaco-
poeias as one of the quality control methods due
to its easiness in handling and long-term and
wide application in research. In this regard, the
most important microscopic feature of ‘Dahuang’
described in these pharmacopoeias can be recog-
nized as the distribution of abnormal vascular
bundles (usually called ‘star spots’), which scat-
tered or formed in ring(s) in the pith of rhizome[1-5].
This distinct secondary structure produces xylem
 240
outside and phloem inside and rays radiated from the
centre of the ring towards the outside, which has yet
been recorded as an important feature to discriminate
those counterfeits and even three officially recognized
species in many literatures[1-8]. Among other helpful but
less important microscopic features recorded in litera-
tures, the mucilage cavity was believed to exist only in
pith of rhizomes and distributed differently in different
species[1,6]. However, these judgments confront chal-
lenges in our preliminary study. Even though similar
studies on the distribution of mucilage cavities on section
of Radix et Rhizoma Rhei has been conducted about 20
years ago[9-12], but why is such a confusion still kept in
some authorized publications[1,6]? Normally, there are
two simple diagnostic methods for authentication of
‘Dahuang’, one uses abnormal vascular bundles as his-
tological feature, and the other uses rhaponticin as the
chemical marker. Neither one seemed effective enough
for differentiating ‘Dahuang’ from its counterfeits by
some reports[7,10]. If so, mucilage cavities can be assistant
histological feature for authentication of this herb or do
differentiate among three species of ‘Dahuang’ as shown
in above reports[9,10]? To clarify all above questions, our
research team re-examined total 22 batches of crude
‘Dahuang’ specimens, of which four were purchased in
Hong Kong and eighteen acquired from the endemic
producing areas in mainland China.
1 Materials and Methods
1.1 Procurement and Identification of Crude
Rhubarb Samples
Eighteen batches of ‘Dahuang’ crude drugs with
plant specimens were collected from the endemic pro-
ducing areas in Mainland China, of which 6 specimens
were authenticated as Rheum palmatum L., 6 as Rheum
tanguticum Maxim. ex Balf., and 6 as Rheum officinale
Baill.[13,14]. The plant specimens were deposited in the
herbarium of Department of Chinese Materia Medica.
Moreover, 4 batches of ‘Dahuang’ crude drugs were pur-
chased in the local market of Hong Kong. The materials
were deposited in Department of Pharmacology, The
University of Hong Kong, Hong Kong, China. All samples
are listed in Table 1, which showed the collection details.
1.2 Apparatus
The slicing process was conducted by using a Leica
RM 2016 Rotary Microtome (Leica Instruments Ltd.,
Shanghai, China). The section features were examined
and imagined using Axiophot Universal Microscope
TANG Jun et al : Identification of Mucilage Cavity as a Significant Ă
(Zeiss Group, Germany) and SPOT RTcolor Digital
Camera (Diagnostic Instruments, USA). The SPOT
Advanced software was used for photographing (Diag-
nostic Instruments, USA).
1.3 Chemicals
The pure ethanol and xylene were in analytical
grade and purchased from E. Merck Co.. Canada Balata
(for sealing) and paraplast plus (m.p. 56 ć, for paraffin-
embedding) were the products of Sigma Co.. Both
Safranine O and Fast Green (for staining) were pur-
chased from International Laboratory in USA. For stain-
ing, 1% Safranine O solution in 50% ethanol and 0.5%
Fast Green solution in 95% ethanol were prepared. Other
chemicals for microscopic chemical tests or other uses
were all of analytical grade and the test solutions were
prepared according to procedures described in Appendix
II of the Chinese Pharmacopoeia [1].
1.4 Procedures
The transversal sections of the ‘Dahuang’ specimens
were prepared with conventional methods for paraffin
sections[15]. For observation of cell contents, sections
were also prepared by free hand. The staining was con-
ducted with double staining method with safranine O and
fast green. The sections were commonly cropped in the
middle part of the rhizomes and roots. Microscopic
measurement was conducted by means of a micrometer
eyepiece. All slides of ‘Dahuang’ samples were prepared
and examined in triplicate. As for microscopic measure-
ment, at least 20 data were collected for each feature for
calculation.
2 Results and Discussion
There are about 40 species of Rheum plants distrib-
uted in China but only three species under section Pal-
mata can be used for the officially recognized medicine[7].
In this study, 18 batches of the ‘Dahuang’ samples (6
batches for each species) were collected from Mainland
China, of which each represents different geographical
origin, such as Qinghai, Ningxia, Sichuan, Chongqing,
Shannxi, Yunnan and Guizhou. Moreover, 4 batches of
samples were locally bought. Their macroscopic descrip-
tion and transverse section were thoroughly examined
with above-mentioned methodologies, and their typical
features useful for identification of this multi-species
herb drug and descriptions are summarized in Table 1
and shown in Fig.1-4.
2.1 About Macroscopic Identification
In view of the original plants, difference can be
Wuhan Univ. J. Nat. Sci. 2008, Vol.13 No.2 241

clearly found among these three species. However, being another. The varieties in general appearance seem the
a crude drug, as per the morphologic characters, the three same among the different batches of samples even dif-
different species seem to have little difference from one ferent species (data not shown here). At least we could

Table 1 Collection details and distribution of features on transverse section of Radix et Rhizoma Rhei
Mucilage cavities
Species or Batch Plant producing Plant collection Star spots in
Phloem
sources number area year rhizome
Rhizome Star spots Root
Rheum DH-04-01 Xining, Qinghai 2002 in rings or
Present Present Present
palmatum scattered
DH-11-01 A’ba, Sichuan 2003 in a ring or
Present Present N.D.
scattered
DH-14-01 Huzhu, Qinghai 2003 scattered Absent Absent Present
DH-12-01 Ledu, Qinghai 2003 scattered or
Absent Absent Present
in a ring
DH-13-01 Guoluobanma, 2003 scattered or
Present Present Present
Qinghai in a ring
DH-21-01 Ledu, Qinghai 2004 scattered or Partially
Present Present
in a ring present
Rheum DH-08-01 Maixiu, Qinghai 2003 scattered or
Present Present Present
tanguticum in a ring
DH-15-01 Guoluo, Qinghai 2003 in a ring or Present,
Present N.D.
scattered in 4 rings
DH-17-01 Tongren, 2003 in a ring or
Present Present Present
Qinghai scattered
DH-16-01 Huangnan Zhou, 2003 in a ring or
Absent Present Present
Qinghai scattered
DH-24-01 Huangnan Zhou, 2004 in a ring or
Present Present Present
Qinghai scattered
DH-25-01 Guyuan, Ningxia 2004 in a ring or Partially Partially
Present
(wild) scattered present present
Rheum DH-05-01 Beichuan, 2002 in a ring or
Absent Absent N.D.
officinale Sichuan scattered
DH-18-01 Nanchuan, 2003 in rings or
Absent Absent N.D.
Chongqing scattered
DH-19-01 Dali, Yunnan 2003 In a ring or more
Present Present Present
or scattered
DH-20-01 Beichuan, 2003 in a ring or Present,
Present N.D.
Sichuan scattered in 2-5 rings
DH-22-01 Zunyi, Guizhou 2004 in a ring or more
Absent Absent N.D.
or scattered
DH-23-01 Hanzhong, Shanxi 2004 in a ring or more
Absent Absent N.D.
or scattered
From local L-DH-1 Gansu* in a ring or
Present Present N.D.
market scattered
L-DH-2 Gansu* in a ring or
Present Present N.D.
scattered
*
L-DH-3 Gansu in a ring or
Present Present N.D.
scattered
L-DH-4 Sichuan* in rings or
Present Present Present
scattered

Commercially proposed; N.D.: Not Determined
 242
not distinguish R. palmatum and R. tanguticum from one
another. This maybe due to their close taxonomic status
(R. tanguticum is a variation of R. palmatum). Only a
little difference may be found between R. officinale and
the above-mentioned species, because some samples in
almost all batches of R. officinale are in typical hip-
pocrepiform shape in appearance but never the case in
other two species. R. officinale is usually called ‘hip-
pocrepiform Dahuang’ not only due to the geographical
distribution but also the distinct shape in appearance
(Fig.1).
Fig.1 A representative photograph of Radix
et Rhizoma Rhei
A. Rhizome; B. Root
1. Rheum palmatum L.; 2. Rheum tanguticum Maxim. ex Balf.; 3. Rheum offi-
cinale Baill.
Moreover, the rhizomes and roots of ‘Dahuang’
being traded in the Hong Kong market have in different
shapes, which basically are the same for each species, for
which the reason may depend more on the plants’ geo-
graphical origin or different preliminary treatment on the
spot of collection than on the different species. For
TANG Jun et al : Identification of Mucilage Cavity as a Significant Ă
example, 4 local samples (L-DH-1 to L-DH-4) were
selected in our study and authenticated as the real
‘Dahuang’ (Table 1); even though we can probably dif-
ferentiate ‘Dahuang’ derived from R. officinale with those
from other two species according to the above-mentioned
feature in shape, it is still difficult for us to exactly know
which sample is under which species only through the
general appearance such as in subcylindrical or conical.
Furthermore, the variations in appearance lead to
immediate requirement of changing the ranges of length
and diameter of the rhizomes and roots also because the
current data of the Chinese Pharmacopoeia may have
been existing for many years[1,16].
2.2 About Microscopic Identification
The results of microscopic examination usually
come from the powder and section of the rhizome or root.
Even though many microscopic characteristics have been
studied or described in many previous publications, from
which we can still find some confusion. Most of note-
worthy in our study is about microscopic specification of
the mucilage cavities, one feature on transverse section
of ‘Dahuang’ (Table 1).The results show that the muci-
lage cavities often exist in the phloem of rhizome, root or
abnormal vascular bundles. Even though this finding has
been reported many years before[9,10], its description in
the pith of rhizome are still specified as ‘pith of rhizome
broad, usually showing mucilage cavities’ in current
Chinese Pharmacopoeia or other literature[1,6]. This may
be misleading unless the mucilage cavities of abnormal
vascular bundles are present in pith. Our study should be
a further verification of the former pharmacognostic
studies on ‘Dahuang’. Moreover, as a microscopic fea-
ture, the mucilage cavities are obvious and easily recog-
nized after double staining and can be found in 4 batches
of Rheum palmatum, almost all 6 batches of R.
tanguticum and 2 batches of R. officinale in our study.
The contents inside (mucilage) are shown to be trans-
parent when un-colored and reddish-brown with sa-
franine and fast green in double staining existing. The
similar arrangement of the mucilage cavities in phloem
can be found in almost all the three species, that is,
mostly scattered or in a discontinuous concentric ring,
sometimes in 2-5 rings (Fig.2-4). We think that it is not
distinct enough to realize the differentiation of three offi-
cial species of ‘Dahuang’ as shown in above-mentioned
studies[9,10]. In these studies, however, three species can
be distinguished from each other by the presence or ab-
sence of mucilage cavities or the amount of mucilage
cavities[9], for example, absent in Rhei officinalis Radix
Wuhan Univ. J. Nat. Sci. 2008, Vol.13 No.2 243

Fig.2 Microscopic features of transverse section of Rhizome of Rheum palmatum

1. Cork; 2. Cortex; 3. Cleft; 4. Phloem; 5. Phloem ray; 6. Mucilage cavities; 7. Cambium; 8. Xylem; 9. Xylem ray; 10. Vessels; 11. Pith; 12. Ray in anomalous vascular
bundle; 13. Xylem of anomalous vascular bundle; 14. Cambium of anomalous vascular bundle; 15. Phloem of anomalous vascular bundle

et Rhizoma (‘Yaoyong Dahuang’), present in Rhei pal-
mate Radix et Rhizoma (‘Zhangye Dahuang’), and many
exist in Rhei tangutici Radix et Rhizoma (‘Tanggute
Dahuang’)[9]. The reason for our different results may be
the different geographic sources, changed growing
characteristics, collection time, and amounts of speci-
mens available. Furthermore, it seems also unsuitable to
differentiate them from the unofficial ‘Dahuang’ because
mucilage cavities may also be found in their roots or
rhizomes as shown in our data and previous report[10],
but it could be useful if only associated with other fea-
tures. Further studies will be conducted on its real status
in the herb identification and evaluation when more
unofficially recognized counterfeits can be collected.
As regards the authentication and evaluation of the
official ‘Dahuang’, it is interesting to mention the pres-
ence and distribution of abnormal vascular bundles, an
important characteristic presents in its rhizomes[1-5,9-12].
 244 TANG Jun et al : Identification of Mucilage Cavity as a Significant Ă

Fig.3 Microscopic features of transverse section of Rhizome of Rheum tanguticum

1. Phloem; 2. Phloem ray; 3. Mucilage cavities; 4. Cleft; 5. Cambium; 6. Xylem; 7. Xylem ray; 8. Vessels; 9. Pith; 10. Xylem of anomalous vascular bundle; 11. Rays
in anomalous vascular bundle; 12. Cambium of anomalous vascular bundle; 13. Phloem of anomalous vascular bundle; 14. Mucilage cavities in phloem of anomalous
vascular bundle

Abnormal vascular bundles represent a special secondary
structure in rhizomes by showing phloem inside and
xylem outside, and the rays running radiately from the
centre of the ring towards the outside forming whirls of
tissues. So far, no counterfeit of the official ‘Dahuang’ is
reported containing this feature except for one unofficial
‘Dahuang’ derived from Rheum emodi of the section
Rhapontica[6, 9-12]. However, the characteristic of abnor-
mal vascular bundles in this species could be distin-
guished by fewer in number on transverse section, and
bigger in size[12]. Moreover, some internal variations
were observed in those with different geological origins
and resulted in the conflicting reports[6,12]; and the trans-
versely cut surface of this species showed a bright bluish
violet fluorescence under ultraviolet light, indicating the
rhaponticin content[7,12]. Its taxonomical status (maybe
hybrid origin or not) therefore needs further investiga-
tion[11]. Further to the official species, we find that there
Wuhan Univ. J. Nat. Sci. 2008, Vol.13 No.2 245

Fig.4 Microscopic features of transverse section of Rhizome of Rheum officinale

1. Cork; 2. Cortex; 3. Cleft; 4. Phloem; 5. Phloem ray; 6. Mucilage cavities; 7. Cambium; 8. Xylem ray; 9. Xylem; 10. Vessels; 11. Pith; 12. Xylem of anomalous vas-
cular bundle; 13. Rays in anomalous vascular bundle; 14. Cambium of anomalous vascular bundle; 15. Phloem of anomalous vascular bundle; 16. Mucilage cavities in
phloem of anomalous vascular bundle

are some different descriptions or even confusions for
the characteristic of abnormal vascular bundles in many
national pharmacopoeias. For example, the feature
existed as a ring inside the pith of rhizome[3,4]; scat-
tered[1,2]; or scattered in R. officinale but arranged in a
ring in R. palmatum[5]. There was also study to indicate
that the arrangement of abnormal vascular bundles inside
the pith was almost the same in all three species, that is,
in rings or a ring and scattered, seem to run down from the
top to the end of rhizomes[6,11]. Our finding is compatible
with this conclusion. The microscopic characteristics of
abnormal vascular bundles in transverse section are found
to be almost the same in all examined authenticated sam-
ples from all the three species of ‘Dahuang’, as described
in Table 1. These results support the fact that the abnormal
vascular bundles can be considered as a distinct feature for
 246
identification of a true ‘Dahuang’ but not a feature for dis-
crimination of three species[6].
In comparison with chemical purity test, we think
more confidently that the abnormal vascular bundles, as
a biologically inherited characteristic, should be impor-
tant in authentication of the official ‘Dahuang’. This is
because there are some limits about using rhaponticin for
purity test. Some researchers found rhaponticin also
contained in trace in some authentic ‘Dahuang’ samples,
or absent in some adulterants of ‘Dahuang’, such as in
some Rumex species, which were usually known as
‘Tu-Dahuang’ (native rhubarb) used in folk medicine[7].
3 Conclusion
In conclusion, our detailed studies on the macro-
scopic and microscopic characteristics of Radix et Rhi-
zoma Rhei have led to the following points which were
not delineated clearly and harmonized in numerous cur-
rent publications and even national standards:ķthe
mucilage cavities did exist in phloem of the rhizome and
root or abnormal vascular bundles but not the pith of
Radix et Rhizoma Rhei, and the characteristics in three
official species of ‘Dahuang’ were not distinct enough to
differentiate each other by their similar distribution;
ĸabnormal vascular bundles, as a biologically inher-
ited feature in pith of rhizome, which presence and ar-
rangement on transverse section can be used as an au-
thentic characteristic of Radix et Rhizoma Rhei but still
not effective for differentiation of three species;Ĺ the
appearance of Radix et Rhizoma Rhei was varied, which
may more depend on the plants’ geographical origin or
different preliminary treatment on the spot of collection
than on the species difference. These results strongly
suggested that the microscopic or macroscopic descrip-
tions of Radix et Rhizoma Rhei should be modified and
updated in the Chinese Pharmacopoeia or other interna-
tional Pharmacopoeias.
Acknowledgements: The authors are sincerely
grateful to Professor Kwok-Fai So, Department of
Anatomy, Faculty of Medicine, The University of Hong
Kong for his kindness on providing precious research
equipments.
References
[1] State Pharmacopoeia Commission of the P. R. China. Phar-
macopoeia of the People’s Republic of China (Chinese Edi-
TANG Jun et al : Identification of Mucilage Cavity as a Significant Ă
tion 2005) [M]. Beijing: Chemical Industry Press, 2005:
17-18(Ch).
[2] Society of Japanese Pharmacopoeia. Japanese Pharmaco-
poeia (English version) [M]. 14th ed. Tokyo: Junkudo Book
Shop, 2001: 1026-1027.
[3] European Pharmacopoeia Commission. European Pharmaco-
poeia [M]. 4th ed. Strasbourg: Council of Europe, 2002: 291.
[4] General Medical Council. The British Pharmacopoeia [M].
London: Constable & Co., England, 2003: 1620.
[5] World Health Organization. WHO Monographs on Selected
Medicinal Plants [M]. Geneva: World Health Organization,
Dept. of Essential Drugs and Medicines Policy, 2004: 231- 240.
[6] Xiao Peigen. Modern Chinese Materia Medica, Vol. 1[M].
Beijing: Chemical Industry Press, 2002: 66-76 (Ch).
[7] Xiang Lan, Yang Meihua, Fan Guoqiang, et al. A Preliminary
Study of Chemotaxonomy of Rheum Species [J]. Journal of
Beijing Medical University, 1998, (Suppl 6): 57-59 (Ch).
[8] Xu Guojun, Xu Luoshan. The Chinese Materia Medica, Vol.
1[M]. Beijing: China Medicinal Science and Technology
Press, 1996: 130-136 (Ch).
[9] Lou Zhicen, Wang Xuan, Masayuki M, et al. Pharmacognos-
tical Studies on the Chinese Crude Drug “Da-Huang, Rhu-
barb” (I) Botanical Origin of the Official Da-Huang [J].
Japanese Journal of Pharmacognosy, 1988, 42: 291-301.
[10] Wang Xuan, Lou Zhicen, Masayuki M, et al. Pharmacognos-
tical Studies on the Chinese Crude Drug “Da-Huang, Rhu-
barb” (II) Botanical Origin of Three Unofficial Da-Huang [J].
Japanese Journal of Pharmacognosy, 1988, 42: 302-309.
[11] Lou Zhicen, Wang Xuan, Masayuki M, et al. Pharmacognos-
tical Studies on the Chinese Crude Drug Da-Huang IV, on the
Abnormal Vascular System and Coil-Like Vessel in the Rhi-
zomes of Certain Rheum spp [J]. Journal of Japanese Botany,
1989, 64: 97-107.
[12] Tsuneo N, Wang Xuan, Masayuki M, et al. Pharmacognosti-
cal Studies of Chinese Crude Drug Da-Huang (Rhubarb) (ċ)
Some Rheum species Used as Chinese Folk Medicine (1) [J].
Japanese Journal of Pharmacognosy, 1989, 43: 109-120.
[13] Feng Yibin, Tong Yao, Tang Jun, et al. Standardization of
Chinese Materia Medica in Hong Kong [J]. Shanghai Journal
of Traditional Chinese Medicine, 2005, 39: 25-30 (Ch).
[14] Li Anren, Gao Zuojing, Mao Zumei, et al. Flora of China Vol.
25 [M]. Beijing: Science Press, 1991: 182- 185 (Ch).
[15] Wang Yongzhen. Experimental Guideline for Identification of
Chinese Materia Medica [M]. Shanghai: Shanghai Science
and Technology Press, 1998: 31-33 (Ch).
[16] Xu Guojun, Shi Dawen. Pharmacogonosy [M]. Beijing: Peo-
ple’s Health Press, 1986: 162-166 (Ch).
ƶ