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: aryakr@rediffmail.com, sayyadak@yahoo.com; sayyadak@nbri.res.in

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

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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.

References 1 Arya KR & Agarwal SC, Folk therapy for eczema, bone

fracture, boils and gingivitis in Taragtal province of Uttaranchal, Indian J Tradit Knowle, 7 (2008) 443-145.

2 Arya KR, Sharma D & Kumar B, Validation and quality determination of an ethnobotanical lead for osteogenic activity isolated from Ulmus wallichiana Planch.: A traditional plant for healing fractured bones, J Sci Ind Res, 70

(2011) 360-364. 3 Anonymous, IUCN Red list of threatened species, 2006.

(www.iucnredlist.org) 4 Phartyal SS, Thapaliyal RC & Nayal JS, Ulmus walliciana

(elm)-An endangered tree of economic value, MFP News, 7 (1997) 1 8-19.

5 Samant SS, Dhar U & Palani LMS, Medicinal Plants

of Indian Himalaya, (Gyanodaya Parkashan, Nainital,

Uttarakhand), 1999. 6 Gaur RD, Flora of District Garhwal, North West Himalaya,

(Trans Media, Srinagar, Garhwal, India), 1999. 7 Rawat P, Kumar M, Sharan K, Chattopadhyay N &

Maurya R, Ulmosides A and B: Flavonoid 6-C- glycosides from Ulmus wallichiana, stimulating osteoblast differentiation assessed by alkaline phosphatise, Bioorganic Med Chem Let, 19 (2009) 4684-4686.

8 Maurya R, Rawat P, Sharan K, Siddiqui JA, Swarnkar G, Manickavasagam L, Arya KR & Chattopadhayay N, Novel flavonol compounds, A bioactive extract/fraction from

Ulmus wallichiana and its compounds for prevention for treatment of Osteo -health related disorders, US Patent

110003, 11, September 2009. (www.wipo.int/pctb/en/wo.jsp? WO=2009110003)

9 Sharan K, Siddiqui JA, Swarnkar G, Tyagi AM, Kumar A, Rawat P, Kumar M, Nagar G, Arya KR, Manickavasagam L, Jain G, Maurya R & Chattopadhyay N, Extract and fraction from Ulmus wallichiana Planchon promotes peak bone achievement and have a nonestrogenic osteoprotective effect, Menopause 17 (2010) 393-402.

10 Khatoon S, Singh N, Srivastava N, Rawat AKS & Mehrotra S, Chemical evaluation of seven Terminalia species and quantification of important polyphenols using HPTLC, J Planar Chromatogr, 21 (3) (2008) 167–171.

11 Khatoon S, Rai V, Rawat AKS & Mehrotra S, Comparative pharmacognostic studies of three Phyllanthus species, J Ethnopharmacol, 104 (2006) 79-86.

12 Khatoon S, Srivastava M, Rawat AKS & Mehrotra S,

HPTLC method for chemical standardization of Sida

species and estimation of the alkaloid ephedrine, J Planar

Chromatogr, 18 (2005) 364-367. 13 Khatoon S & Mehrotra S, Bark Drugs, Vol 1, (National

Institute of Science Communication and Information Resources, Council for Scientific and Industrial Research, New Delhi), 2009.

14 Brain KR & Turner TD, The Practical Evaluation

of Phytopharmaceuticals, (Wright-Scientechnica, Bristol), 1975, 36-45.

15 Kokate CK, Practical Pharmacognosy, Ist edn, (Vallabh Prakashan, New Delhi), 1986, 111.

16 WHO/QCMMPM, Quality Control Methods for Medicinal

Plant Material, (Organisation Mondiale De La Sante, Geneva), 1992, 22-34.

17 Khatoon S, Singh N, Kumar S, Srivastava N, Rathi A & Mehrotra S, Authentication and quality evaluation of an

important Ayurvedic drug - ‘Ashoka Bark’, J Sci Ind Res, 68

(2009) 393-400. 18 Wagner H & Bladt S, Plant Drug Analysis, 2nd edn,

(Springer-Verlag Berlin Heidelberg, New York), 1984.