Indian Journal of Traditional Knowledge Vol. 12 (4), October 2013, pp. 664-669
Development of quality control markers for Ulmus wallichiana Planchon:
An Indian traditional plant for osteogenic activity
K R Arya1*, Sayyada Khatoon2 & Brijesh Kumar3 1Botany Division, 3Sophisticated Analytical Instrument Facility CSIR- Central Drug Research Institute, Lucknow- 226001, India,
2Pharmacognosy & Ethnopharmacology Division, CSIR- National Botanical Research Institute, Lucknow- 226 001, India E-mails: [email protected], [email protected]; [email protected]
Received 17.01.12, revised 31.05.13
Ulmus wallichiana Planchon, is an Indian folk traditional plant used for the treatment of fractured bones in folklore tradition of Uttarakhand Himalaya, India. During chemical investigations, three major compounds, Ulmoside A & B, and Naringenin-6-C-β-D glucopyranosideis for osteogenic activity have been isolated from bark of this plant species. This paper presents macro and microscopic study, physicochemical parameters and chemo-profiling of stem bark of U. wallichiana through Q TOF HRMS for development of quality control markers and identification of crude samples. Transverse section (TS) stem bark and powder microscopy shows crushed rhytidoma consisting plenty of mucilaginous canals followed by crushed cork, cells of the cork filled with dark brown content. Whereas, authenticity and quality of raw materials can be determinate with exact calculated mass value of marker compounds for osteogenic activity at 467, 451 and 435 using Q TOF
HRMS technique. Ursolic acid, β-sitosterol and lupeol at Rf value 0.25, 0.36 and 0.44 has also identified as HPTLC marker for identification and authentication of crude drug samples of stem bark of U. wallichiana.
Keywords: Ulmus wallichiana, Bone healing, Q TOF HRMS, Quality control marker, HPTLC
IPC Int. Cl.8: A61K 36/00, A01D 20/00, A01D 9/02, G07C 3/14, A62B
Himalayan elm, Ulmus wallichiana Planchon,
family Ulmaceae, locally known as Chamourmou is commonly used for healing fractured bones in
animal as well as human being in folk tradition
of Uttarakhand Himalaya, India1-2
. Plant species is
rare3-4
and endemic to Western Himalaya5. Habitat
degradation, biotic interferences and unsustainable
harvesting of bark shows decline in species richness
and total tree density in Western Himalaya, India. Globally, about 45 species of this genus is found
in North temperate region, while 3 species, i.e.
U wallichiana, U. lancefolia and U. parviflora
are reported from India6. Two novel nonestrogenic
osteoprotective compounds Ulmoside A and
Ulmoside B rich in C-glycosylated flavanoid
and third known compound Naringenin-6-C-β-D glucopyranosideis isolated from same extract
7
showed peak bone achievement and prevention
of menopausal bone loss in ovariectomized rat and promotes osteoblast function and inhibited
adipogenesis8-9
. During pharmacological investigation
these compounds revealed therapeutic potential for
treatment of post menopausal osteoporosis9 and are
now in public domain for commercial exploration.
Source of raw materials for pharmaceutical production is only from wild. Unprecedented demand
and scarcity of raw materials may lead to chances
of adulteration and substitution of genuine drug. Chemical and biological evaluation, physico-chemical
parameters, HPTLC finger printing is useful for
identification and authentication of genuine drugs and
their quality evaluation10-13
. This study provides macro and microscopic studies of stem bark of
U. wallichiana pertaining with HPTLC profiling of
ethanolic extract, physico-chemical parameters and chemical fingerprinting through Quadrupole-Time-
of-Flight High Resolution Mass Spectrometer
(Q-TOF HRMS) to insure quality and authenticity of crude drugs.
Methodology
Ulmus wallichiana Planchon, large deciduous tree
with rough grey exfoliating bark, up to 30 m high; young branches pubescent to tomentose. Leaves
elliptic-acuminate to obovate-cuspidate,7-15 cm long,
4-6 cm broad, base obliquely cuneate to rounded, —————— * Corresponding author
ARYA et al.: QUALITY CONTROL MARKERS OF OSTEOGENIC ULMUS WALLICHIANA
665
sharply biserrate, teeth arching with 2-4 secondary
teeth, upper surface pubescent to scabridulous,
lower surface densely pubescent to tomentose; petiole 6-10 mm long, pubescent. Inflorescence axis
elongated, pedicels more than 5 mm long, articulated;
1/3rd lower portion of pedicel uniformly pilose;
flowers in clusters appearing before leaves on branches of previous season, perianth tube narrowed
into the pedicel, lobes 5-6, obtuse, pubescent to
sub glabrous; stamens 5-6, filaments longer than the perianth, anthers red; ovary slightly pubescent
all over; fruit samara, orbicular-obovate, 12-15 mm,
narrowed into a short stipe, 2-3 mm long, stipe
longer than the perianth, central seed hirsute to sub glabrous, wing membranous, reticulate, margin
ciliolate (Fig. 1A).
Stem bark of Ulmus wallichiana was collected from three different altitudinal locations
of Uttarakhand Himalaya, viz. Nainital (1800 M),
Almora (2500 M) and Bageshwar (3000 M) during April, 2009. All the samples were properly
identified by one of the senior author (KRA)
and compared with Flora of The District Garhwal,
North West Himalaya6. Herbarium specimens
were deposited in departmental herbarium, CDRI,
Lucknow, vide voucher specimen numbers KRA
24432, KRA 24443, and KRA 24444, respectively. Analytical grade chemicals from SD Fine, Mumbai
(India) and standards (Lupeol, β-sitosterol and
ursolic acid) were procured from Sigma, St. Louis, MO, USA.
Macro and microscopy of bark and powder were
studied13-15
. Air dried plant material was used for
quantitative determination of ash and extractive values
16. Swelling Index was calculated by modifying
WHO method due to high percentage of mucilage.
100 mg powder was kept in 25 ml of water in a glass-Stoppard measuring cylinder; the material
was shaken repeatedly for 2 hrs and then kept
for 24 hrs to settle down. Volume of the mixture (ml)
was measured as swelling Index. Marker compounds Ulmoside A, B and Naringenin-6-β-D glucopyranoside
were identified through Q-TOF HRMS fitted with
an electro spray (ESI) interface. Analyses utilized the positive ion mode m/z (M+H)
+ for detection of
compounds.
Chromatographic analysis and HPTLC profile
Dried stem bark of U. wallichiana were powdered
and sieved through 44 meshes. Powdered sample (5 gm) was exhaustively extracted with methanol
(4 × 25 mL, each time for 15 min) under reflux on a
water bath at 100°C. Extracts were filtered through
Whatman No. 1 filter paper (separately for each sample), concentrated under reduced pressure, and
lyophilized17-18
. A solution (10 mg mL-1
) of these
extracts and standard solution (0.1 mg mL-1
) of
β-sitosterol, ursolic acid and lupeol were also prepared in methanol. Known quantity of methanolic
extracts along with chemical markers (β-sitosterol,
ursolic acid and lupeol) were applied on to Higlachrosep nano silica UV 254 HPTLC plates of
10x10 cm with 0.2 mm nano silica with fluorescent
indicator (S. D. Fine-Chem. Ltd. India) with the
help of CAMAG Linomat Applicator 5, positioned 15 mm from side and 10 mm from bottom of the
plate. Plate was eluted to a distance of 8.0 cm at
room temperature (24ºC) in a solvent system –toluene: ethyl acetate : formic acid (9 : 1 : 0.1) in
previously saturated twin trough chamber (CAMAG).
Plate was derivatized by spraying with anisaldehyde sulphuric acid reagent. After heating at 110°C for
10 min plate was documented under visible light
and scanned at wavelength 600 nm using CAMAG
TLC Scanner 3 with software Win Cats 3.2.1.
Results and discussion
Macroscopic studies
Bark fibrous, rough grey exfoliating in diamond
shaped flakes. Dried mature bark slightly curved, 1 to 1.5 cm in thickness, outer surface blackish brown
in colour, rough due to the presence of irregular
cracks and rhytidoma which peeled off at some places leaving rough, protuberated brown coloured
surface; internal surface brown in colour, smooth
or with very fine, wavy, transverse and longitudinal
striations; fracture hard and splintery; mucilaginous and slightly acrid in taste with pleasant odour
(Figs.1 B & C).
Microscopic studies
TS bark shows crushed rhytidoma consisting
of plenty mucilaginous canals followed by crushed cork, cells of the cork filled with dark brown
content. Cork cambium is not distinct. Phelloderm is
very broad traversed with plenty schizogenous
mucilage canals, some stone cells, fibres and prismatic crystals of calcium oxalate. Parenchymatous
cells also filled with mucilage. Secondary phloem
is very narrow as compared to phelloderm. Phloem consists of sieve tubes, companion cells, phloem
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Fig. 1—A- Ulmus wallichiana tree; B- Outer surface of stem bark; C- Inner surface of stem bark.
Table 1—Physico-chemical values of U. wallichiana collected from Nainital, Almora and Bageshwar districts in Uttarakhand
S.No. Parameters Nainitala Almoraa Bageshwara
1. Total ash % 15.29 ± 0.315 16.04 ± 0.235 16.23 ± 0.156
2. Acid insoluble ash % 0.94 ± 0.054 0.96 ± 0.016 0.99 ± 0.031
3. Alcohol soluble extract % 22.00 ± 0.100 23.00 ± 0.300 24.30 ± 0.100
4. Swelling Index (mL) 12.50 ± 0.500 13.20 ± 0.300 12.80 ± 0.250
a Mean value of 5 readings ± SD
parenchyma, abundant mucilage canals and tangential
bands of crystalloid fibres interrupted by medullary
rays. Mucilage canals are arranged in tangential and longitudinal rows, forming a checker board
appearance. Frequency of prismatic crystals is more
in phloem region. Medullary rays, heterogenous,
multiseriate, upto 20 cells high, cells circular to polygonal in shape, prismatic crystals of calcium
oxalate abundant in rows (Fig. 2).
Powder
It is reddish brown in colour, mucilaginous
and acrid taste without any odour. Under the microscope it shows plenty mucilaginous canals,
numerous prismatic crystals and some cluster
crystal of calcium oxalate, groups of
parenchymatous cells, crystalloid, septate and non-septate fibres, cork cells in transverse and
surface view and stone cells (Fig. 3).
Physico-chemical analysis
Ash values (total, acid insoluble, water-soluble),
alcohol soluble, and water-soluble extractives are presented in Table 1. Slight variation was observed
in Physico-chemical parameters of all the samples.
For example, ash values and extractive values
were found maximum in Bageshwar sample while mucilage content was found more in Almora sample.
Q-TOF mass spectrum (Fig. 4) indicated that peak
at m/z 467 and 435 could be due to bioactive markers ulmoside A and peak at 451 could be due to
ulmoside B or eriodictyol-6-C-β-D glucopyranoside.
HPTLC fingerprint profiles of all the three samples collected from Nainital, Almora and
Bageshwar districts along with chemical markers
have been developed (Fig. 5). Three HPTLC marker
compounds, viz. ursolic acid, β-sitosterol and lupeol at Rf value 0.25, 0.36 and 0.44, respectively were
observed in all three samples.
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Fig. 2—Microscopic study of stem bark of U. wallichiana, cr, prismatic crystal of calcium oxalate; f, fibre; mc, mucilage canal; mr, medullary rays; sc, stone cells.
The wealth of traditional knowledge system
related to the use of plants is not important to
record this knowledge just to store, but it is important to keep it alive and make available
for future as a unique resource for identification
of novel pharmacological agents. In recent
years, various factors like gradual depletion of natural pockets of indigenous flora and
fauna, decreasing interest shown by traditional
Fig. 4—A, B, C- Mass spectrum fingerprints, calculated mass and molecular structures of marker compounds (Ulmoside A, B & Naringenin-6-C-β-D glucopyranosideis) in stem bark of U. wallichiana determinated through Q-TOF HRMS.
Fig. 3—Powder microscopy. A- Mucilage canals; B- Prismatic crystals; C- Cluster crystals; D- Stone cells; E- Cork cells in surface view; F- Cork cells in transverse view; G- parenchymatous cells embedded with prismatic crystals, H- Parenchymatous cells; I- Non-septate fibre; J- Septate fibre.
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healers in this profession due to less economic gain, natural calamities etc., led to the loss of
huge amount of indigenous knowledge. Due to
gradual loss of this generational traditional knowledge, these herbal texts become increasingly
valuable and need rapid interaction between
scientist and traditional communities. Sharing of
benefits arises from the product development will further encourage their confidence and may
leads more potential pharmacological agents for
therapeutic values.
Conclusion
U. wallichiana is popular for its medicinal property of healing fractured bones in folk tradition
of Uttarakhand Himalaya and some of its traditional
claims have been scientifically validated. However, a well established quality control and identification
parameters are highly essential for crude drug
identification and authentication of this plant species. It is notable that the osteoprotective
compounds ulmoside A, B and naringenin-6-C-β-D
glucopyranoside is isolated from bark of this plant species are very complex in nature and have been
licensed to Kemextree, USA for product
development. From ongoing discussions, it was
revealed that the molecular mass spectrum, exact calculated mass value and molecular ion peaks
at 467, 451 and 435, identified as Ulmoside A, B
and Naringenin-6-C-β-D glucopyranoside through Q-TOF HRMS and macro and microscopic studies,
physico-chemical parameters, high mucilaginous
substances on soaking in water and HPTLC marker compounds (ursolic acid, β-sitosterol and lupeol)
identified in the stem bark may be used as standard
parameters for authentication and quality evaluation
of commercial samples of U. wallichiana during product development stage.
Fig. 5—HPTLC fingerprint profiles of methanolic extract of all the three samples of stem bark of U. wallichiana along with chemical markers. 1- Almora; 2- Bageshwar; 3- Nainital; R1- β-Sitosterol; R2- Ursolic acid; R3- Lupeol
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Acknowledgement
Author (KRA) is thankful to Director CDRI,
Lucknow for encouragement and Dr N Chattopadhyay, Head, Endocrinology Division, for financial support
from MOH project. Department of Forest, Govt of
Uttarakhand and local informants of Taragtal, Almora
and Chaura, Bageshwar, Kumaon are thankful for kind support during ethnobotanical survey and collection of
plant samples.
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