STANDARD ANATOMICAL AND PHYSICOCHEMICAL ATLAS OF …

Mallya et al. European Journal of Biomedical and Pharmaceutical Sciences

www.ejbps.com │ Vol 8, Issue 2, 2021. │ ISO 9001:2015 Certified Journal │

252

STANDARD ANATOMICAL AND PHYSICOCHEMICAL ATLAS OF ASHOKA (SARACA

ASOCA (ROXB.) WILLD.), A BARK USED IN INDIAN SYSTEM OF MEDICINE

B. R. Lalitha1 and Mallya Suma V.

2*

1Professor and Head, Department of Dravyaguna, Government Ayurveda Medical College, Bangalore. 2*Associate Professor, Sri Dharmasthala Manjunatheshwara College of Ayurveda, Kuthpady, Udupi.

Article Received on 20/12/2020 Article Revised on 10/01/2021 Article Accepted on 30/01/2021

INTRODUCTION

Ashoka (Saraca asoca (Roxb.) Willd.) popularly used as

uterine tonic in Ayurveda. This is a medium sized slow

growing tree found naturally as well as in cultivated form

throughout India.[1]

Bark of this tree is used in medicine,

said to be used in infertility, uterine disorder, skin

ailments, painful swellings. Ashokarishta, Ashokadi

ghrita, Tilvaka ghrita, Maha kalyanaka ghrita are few

important formulations.[2]

Tannin, catechin, epicatechin,

Crystalline glycoside, Leucocyanidin, are major

phytochemical constituents reported from the bark.[3]

Flowers are used as styptic in bleeding disorders,

coloring agents as pigmentation disorders. Seeds of this

tree said to be beneficial in calculi.[4]

Because wide

medicinal usage, loss of habitat, and slow growing

pattern tree enlisted as VU(Vulnerable species) as per

IUCN.[5]

Stem bark of Polyalthia longifolia is the

common adulterant of this drug. Because of similar

appearance bark of Bauhinia variegate, Trema orientalis,

Shorea robusta are also said to be admixed.[6]

Efficacy of

a treatment always depend upon quality as well as

novelty of herbal source. Authenticity of herbal material

is major step in drug research.[7]

Hence with all this

background it is planned to take out quality out prints of

this bark through macro-microscopic, physico-chemical

standards and HPTLC.

MATERIALS

Plant materials

Matured bark of Ashoka (Saraca asoca (Roxb.) Willd.)

were collected from their natural habitat during fruiting

season, cleaned properly from extraneous matter,

authenticated using floras and botanists opinion and

sample deposited at SDM center for Research in

Ayurveda and Allied sciences( (V/no. 1095/19021802).

Bark pieces kept in FAA (Formalin-5ml + Acetic acid-

5ml + 70% Ethyl alcohol-90ml) solution for macro-

microscopic study. Few bark pieces shade dried, powder

prepared used for further study.

METHODOLOGY

Macroscopy: External features of bark pieces were

recorded using camera and its colour, shape, size, any

particular smell was recorded.[8]

Microscopy: The bark sample preserved were cut into

thin transverse section using a sharp blade and the

sections stained with saffranine. Transverse sections

photographed using Zeiss AXIO trinocular microscope

attached with Zeiss Axio Cam camera under bright field

light. Magnifications of the figures are indicated by the

scale-bars.[9]

SJIF Impact Factor 6.044 Research Article ejbps, 2021, Volume 8, Issue 2, 252-260.

European Journal of Biomedical AND Pharmaceutical sciences

http://www.ejbps.com

ISSN 2349-8870

Volume: 8

Issue: 2

252-260

Year: 2021

*Corresponding Author: Mallya Suma V. Associate Professor, Sri Dharmasthala Manjunatheshwara College of Ayurveda, Kuthpady, Udupi.

DOI: https://doi.org/10.17605/OSF.IO/RJYPF

ABSTRACT

About: Ashoka (Saraca asoca (Roxb.) Willd.) popularly used as uterine tonic in Ayurveda. Wide medicinal usage

and changed environmental condition made drug obtainable hardly. Many easily available bark drugs found

adulterated with this. Hence it is planned to take standard quality out prints of the bark as per protocol. Materials

and Methods: Matured bark of test drug collected, cleaned properly from extraneous matter. Macro-microscopic,

physico-chemical standards and HPTLC were conducted. Results: Freshly collected bark looks rough, greyish

green on outer surface with warty protuberances of underlying lenticels, having channeled reddish yellow inner

surface. Microscopically bark shows outer many layers of cork cells, inner cortex with polygonal cells, layered

with few stone cells as wells scleranchymatous cells. Phloem fibres filled with prismatic crystals and narrow strips

of medullary rays found in transverse section. Physico-chemical standard and HPTLC photo documentation reveal

about moisture, mineral matter, extractive values. At 254 nm drug has shown 5 peaks, whereas at long UV 366nm

drug shown 7 componentsand at 620nm drug shown 12 peaks.

KEYWORDS: Ashoka (Saraca asoca (Roxb.) Willd.) physico-chemical, HPTLC.

https://en.wikipedia.org/wiki/William_Roxburgh

https://en.wikipedia.org/wiki/Carl_Ludwig_Willdenow











253

Physicochemical standards

Loss on drying at 105oC

10 g of sample was placed in tared evaporating dish. It

was dried at 105˚C for 5 hours in hot air oven and

weighed. The drying was continued until difference

between two successive weights was not more than 0.01

after cooling in desiccator. Percentage of moisture was

calculated with reference to weight of the sample.[10]

Total Ash

2 g of sample was incinerated in a tared platinum

crucible at temperature not exceeding 450˚C until carbon

free ash is obtained. Percentage of ash was calculated

with reference to weight of the sample.

Acid insoluble Ash

To the crucible containing total ash, add 25ml of dilute

HCl and boil. Collect the insoluble matter on ashless

filter paper (Whatmann 41) and wash with hot water

until the filtrate is neutral. Transfer the filter paper

containing the insoluble matter to the original crucible,

dry on a hot plate and ignite to constant weight. Allow

the residue to cool in suitable desiccator for 30 mins and

weigh without delay. Calculate the content of acid

insoluble ash with reference to the air dried drug.

Water soluble ash

Boil the ash for 5 min with 25 ml of water; collect

insoluble matter on an ashless filter paper, wash with hot

water, and ignite for 15 min at a temperature not

exceeding 450˚C. Subtract the weight of the insoluble

matter from the weight of the ash; the difference in

weight represents the water soluble ash with reference to

the air-dried sample.

Alcohol soluble extractive

Weigh accurately 4 g of the sample in a glass stoppered

flask. Add 100 ml of distilled Alcohol (approximately

95%). Shake occasionally for 6 hours. Allow to stand for

18 hours. Filter rapidly taking care not to lose any

solvent. Pipette out 25ml of the filtrate in a pre-weighed

100 ml beaker. Evaporate to dryness on a water bath.

Keep it in an air oven at 105C for 6 hours, cool in

desiccator for 30 minutes and weigh. Calculate the

percentage of Alcohol extractable matter of the sample.

Repeat the experiment twice, and take the average value.

Water soluble extractive:

Weigh accurately 4 g of the sample in a glass stoppered

flask. Add 100 ml of distilled water, shake occasionally

for 6 hours. Allow to stand for 18 hours. Filter rapidly

taking care not to lose any solvent. Pipette out 25ml of

the filtrate in a pre-weighed 100 ml beaker. Evaporate to

dryness on a water bath. Keep it in an air oven at 105C

for 6 hours. Cool in a desiccator and weigh. Repeat the

experiment twice. Take the average value.

HPTLC 1g of bark of Ashoka powder was suspended in 10 ml of

alcohol warmed on a water bath and filtered. 4, 8 and

12µl of the above extract was applied on a pre-coated

silica gel F254 on aluminum plates to a band width of 7

mm using Linomat 5 TLC applicator. The plate was

developed in Toluene: Ethyl acetate: Formic acid:

Methanol (3.0: 3.0: 0.8: 0.2). The developed plates were

visualized under short UV, long UV and then derivatised

with vanillin sulphuric acid and scanned under UV

254nm, 366nm. Rf, colour of the spots and densitometric

scan were recorded.[11]

RESULTS

Macroscopy

Freshly collected bark rough, greyish green on outer

surface with warty protuberances of underlying lenticels.

Inner surface channeled reddish yellow in colour with

astringent taste. Fracture fibrous without having

particular smell. (Figure 1).

Outer surface



254

Inner surface

Figure 1. Macroscopy of Bark of Ashoka(Saraca asoca (Roxb.) Willd.)

Microscopy

Transverse section of the bark shows the presence of

outer thirty or more rows cork cells. Outermost cork cells

are much compressed and cell walls are wavy, whereas

innermost are rectangular. Cortex composed of thin

walled polygonal cells with small intercellular spaces

and several prominent groups of stone cells. Hundred

thick bands of stone cells called sclerides found below

this layer. Few parenchymatous cells contain starch

grains inner to sclerenchymatous cells. Phloem fibres

embedded with prismatic crystals found in this cortex

layer. Medullary rays are narrow, uni-biseriate in the bast

but broaden much towards their distal ends. (Figure 2)

Fig 2 a. T.S of Bark





255

Fig 2b. A portion of bark enlarged.

Figure 2: Microscopy of bark of Ashoka (Saraca asoca (Roxb.) Willd.)

Ca – cambium; Ck – cork; Ct – cortex; MR – medullary rays; PF – pericyclic fibres; Ph – phloem; Prc – Prismtic

crystals of calcium oxalate; Scl – sclereids;SC – stone cell.

Fig 2c. Cork and Cortex

Ck→

Ct→

Pa→

Scl→

SG→

SC→

Pg→





256

Fig 2 d. Phloem

Ck – cork; Ct – cortex; MR – medullary rays; PF – pericyclic fibres; Pg – phellogen; Ph – phloem; Prc – Prismtic

crystals of calcium oxalate; Scl – sclereids;SC – stone cell; SG – starch grains.

Fig 2 e. Fig 2 f. Band of sclereids

MR→

PF→

Prc→

Ck→

Ct→

Pa→

SC→

Pg→

Scl→ Scl→

SG→ Scl→



257

Fig 2 g.Parenchyma containing starch Fig 2 h.Phloem, Phlem fibres embedded with

prismatic crystals.

Ck – cork; Ct – cortex; MR – medullary rays; PF – pericyclic fibres; Pg – phellogen; Ph – phloem; Prc – Prismtic

crystals of calcium oxalate; Scl – sclereids;SC – stone cell; SG – starch grains.

Physicochemical standards

Bark powder of test drug showed following values when

tested as per standard protocol. Loss on drying found as

3.47±0.02, whereas total ash was 7.54±0.06. Acid

insoluble ash was 0.76±0.14 and water soluble ash

was3.78±2.39. Extractive values have shown 12.13±0.02

and 13.95±0.05 in alchohol and water media. (Table 1).

Table 1: Results of standardization parameters of Bark of Ashoka ( Saraca asoca (Roxb.) Willd.).

Parameter Results n = 3 %w/w

Loss on drying 3.47±0.02

Total Ash 7.54±0.06

Acid Insoluble Ash 0.76±0.14

Water soluble Ash 3.78±2.39

Alcohol soluble extractive value 12.13±0.02

Water soluble extractive value 13.95±0.05

HPTLC

Ethanolic extract of test drug powder applied 3, 6 and

9µl on a pre-coated silica gel F254 on aluminum plates

using Linomat 5 TLC applicator using solvent system.

HPTLC photo documentation of developed plates were

visualized under short UV, long UV and then derivatised

with vanillin sulphuric acid and scanned under UV

254nm, 366nm (Figure 3, Table 2). Rf, colour of the

spots and densitometric scan were recorded shown in

Table. At 254 nm drug has shown 5 peaks, whereas at

long UV 366nm drug shown 7 components. After post

derivatization 620nm drug shown 12 peaks. (Figure 4).

Prc→

SG→

PF→

MR→

Pa→

Pa→





258

Short UV Long UV After derivatisation

Figure 3. HPTLC photo documentation of ethanol extract of Bark of Ashoka( Saraca asoca (Roxb.) Willd.)

Solvent system - Toluene: Ethyl Acetate: Formic acid: Methanol (3.0:3.0:0.8:0.2)

Track 1 – Bark of Ashoka– 3µl



Table 2: Rf values of sample of Bark of Ashoka( Saraca asoca (Roxb.) Willd.)

Short UV Long UV After derivatisation

0.05 (Green) - -

- 0.07 (F. blue) -

- 0.17 (F. blue) -

- - 0.26 (Purple)

0.28 (L. green) 0.28 (F. blue) -

0.36 (L. green) 0.35 (F. green) -

0.43 (L. green) 0.43 (F. blue) -

- - 0.52 (Purple)

0.54 (L. green) 0.54 (F. blue) -

- 0.71 (F. green) -

- - 0.74 (Purple)

- - 0.82 (Purple)

*D-dark; L-light;F-fluorescent

Fig 4a. At 254nm







259

Fig 4b. At 366nm

Fig 4c. At 620nm

Figure 4. Densitometric scan of Bark of Ashoka (Saraca asoca (Roxb.) Willd.).

DISCUSSION

Ashoka (Saraca asoca (Roxb.) Willd.) bark of which is

used in infertility, uterine disorder, skin ailments,

painful swellings in Ayurveda. Because of over

exploitation, and slow growing pattern of this tree

resulted in the stage of extinct. Many bark drugs are said

to be used as adulterant in place of this drug. Authentic

quality of a drug forms a major role in success of a

treatment. Macro-microscopic structure of a drug reveals

its basic anatomical features.[12]

Freshly collected bark

rough, greyish green on outer surface with warty

protuberances of underlying lenticels, having channeled

reddish yellow inner surface. Microscopically bark

shows outer many layers of cork cells, inner cortex with

polygonal cells, layered with few stone cells as wells

scleranchymatous cells. Starch grain filled cells found

inner to cortex. Phloem fibres filled with prismatic

crystals found in this layer. Narrow strips of medullary

rays found in transverse section.







260

Physico-chemical standard of a drug are constants of its

moisture content, ash value, extractive value, presence of

minerals. Loss on drying found as 3.47±0.02, indicative

of its less moisture content. Total ash was detected as

7.54±0.06, denotes more of carbonaceous matter. Acid

insoluble ash was 0.76±0.14, denote few insoluble

mineral matters. Water soluble ash found to be

3.78±2.39. Test drug noticed more soluble in water

(13.95±0.05) than alchohol (12.13±0.02) media.

HPTLC photo documentation of developed plates s of

ethanolic extract of bark of Ashoka were visualized

under short UV, long UV and then derivatized with

vanillin sulphuric acid and scanned under UV 254nm,

366nm (Figure 2). Different chemical components were

detected at various wavelength. At 254 nm drug has

shown 5 peaks, whereas at long UV 366nm drug shown

7 components. After post derivatization 620nm drug

shown 12 peaks.

CONCLUSION

Revival of interest in natural products made essential to

generate chemical, anatomical standard features of these

sample. Ashoka (Saraca asoca (Roxb.) Willd.) widely

used bark drug in Indian system of medicine in

gynecological disorder. Overexploitation of this bark

drug made it commercially available other similar

looking bark drugs. Standard anatomical, physico-

chemical and HPTLC documentation of this drug

features portrayed in this paper help in drug

authentication.

Financial support and sponsorship

Part of the observations is made from findings of a major

research project entitled “Identification of Ashoka

through development of SCAR- An analytical study,

RGU:R&D:A 602, sanctioned by Rajiv Gandhi

University of Health Sciences, Bangalore.

REFERENCES

1. AA Borokar, TA Pansare, Plant profile,

phytochemistry and pharmacology of

Ashoka(Saraca Ashoka(Roxb.), De. Wilde)-A

comprehensive review; International journal of

Ayurvedic and Herbal medicine, 2017; 7(2):

2524-41.

2. Anonymous. The wealth of India, A dictionary of

Indian raw materials & industrial products Vol-IV.

Newdelhi: Council of scientific & industrial

research, 2009; 270-275.

3. Khare CP. Indian Medicinal Plants an illustrated

Dictionary, New Delhi, Springer, 2007; 76.

4. Kirtikar KR and Basu BD, Indian Medicinal plants

Vol-3, Dehradun; International Book Distributors,

1996; 1896.

5. GR Smitha, V Thondaiman; Reproductive biology

and breeding system of Sraca asoca(Roxb.) De

Wilde; a vulnerable medicinal plant; Springer plus,

2016; 5: 2025.

6. YK Sarin, illustrated manual of herbal drugs used in

Ayurveda; Council of Scientific and Industrial

research, New Delhi, 1996; 120.

7. Mallya Suma V, Suchitra Prabhu, Vishwanatha U,

KN Sunilkumar; Anatomical atlas of Panchavalkala-

Effective healing of five bark drugs used in

gynaecological disorder; Journal of Ayurvedic and

herbal medicine 2018; 4(1): 6-13.

8. Bani Shashikala, Mallya Suma V, Prabhu Suchitra,

Quality control constraints of Guozotia abyssynica

Cass. Source of medicinally used edible oil seeds;

The Journal of Phytopharmacology, 2018; 7(5):

431-436.

9. Mallya Suma V, Nesari Tanuja; Antibacterial

activity profile and quality standards of

Cymbopogon citratus Stapf- an aromatic grass used

in Indian system of medicine; Journal of Ayurvedic

and Herbal Medicine, 2016; 2(3): 63-66.

10. Hima Shashidharan, Suma V Mallya, Prabhu

Suchitra, KN Sunilkumar; Identity parameters on

traditionally used antiurolithiatic Herb Scoparia

dulcis Linn.; Journal of Ayurveda and Integrated

Medical Science, Nov- Dec 2017; 2(6): 70-76.

11. Sethi PD, High Performance Thin Layer

Chromatography; Ist Edn. New Delhi, CBS

Publisher and Distributors, 1996; 1-56.

12. Gokhale S B, Kokate CH, Purohit AP. A textbook of

Pharmacognosy, 34th

ed. Pune: Nirali prakashan,

2013; 542-544.



STANDARD ANATOMICAL AND PHYSICOCHEMICAL ATLAS OF …

Documents

Transcript of STANDARD ANATOMICAL AND PHYSICOCHEMICAL ATLAS OF …