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Original Article

A comparative study of histo-pharmacognosy of Chenopodiumalbum Linn. under the impact of Bicycle Industry Effluent

Kavita Tyagi a,*, Sandhya Sharma d, Rajat Rashmi e, Sanjiv Kumar b, Shahidul Khair c

aConsultant (Agro.), National Medicinal Plants Board, Department of AYUSH, Ministry of Health & Family Welfare,

Government of India, INA, New Delhi 110023, IndiabAssistant Director, National Medicinal Plants Board, Department of AYUSH, Ministry of Health & Family Welfare,

Government of India, INA, New Delhi 110023, IndiacResearch Officer, National Medicinal Plants Board, Department of AYUSH, Ministry of Health & Family Welfare,

Government of India, INA, New Delhi 110023, IndiadAssistant Professor, Vidhyavati Mukund Lal Girls (PG) College, Ghaziabad 201001, Uttar Pradesh, IndiaeResearch Officer, Homeopathic Pharmacopoeia Laboratory, Department of AYUSH, Ministry of Health & Family Welfare,

Government of India, Ghaziabad 201001, India

a r t i c l e i n f o

Article history:

Received 29 March 2013

Accepted 27 May 2013

Available online 24 June 2013

Keywords:

Chenopodium album

Histo-pharmacognosy

Industrial effluent

* Corresponding author. Tel.: þ91 (0) 11 2465E-mail address: kavitatyagii@gmail.com (

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.06.002

a b s t r a c t

Aims of the study: To carried out the Histo-Pharmacognosy study of Chenopodium album Linn.

under the influence of Bicycle Industry Effluent.

Method: The industrial effluent was analysed by APHA method. The anatomical studies of

plant were carried out according to Metacalf and Chalk, 1950 were consulted; for chemical

analysis Johanson, 1940, Cromwell, 1955 & Trease and Evans, 1983 were followed. TLC was

analyzed by WHO, 1998.

Results: The physicoechemical parameters of analysed effluent were found higher values

as compared to standard values and morphological & anatomical parameters were found

in decreasing trend in polluted plant samples as compared to non-polluted plant samples.

The colour reaction tests showed only degrees of changes. The number of spots were

decreased in the plant samples of polluted sites. Water and alcohol extractive values were

found to be lowered collected from polluted areas. Ash values were comparatively higher

in polluted plant samples. Stomatal index and Palisade Ratio were lower in polluted leaves.

Vein Islet Number and Vein Termination Number were higher in polluted leaves.

Conclusion: The conclusion of this study is that the plants from non-polluted area should be

collected for quality production of medicines, since majority of parameters reflect

decreasing data values in the plants taken from polluted area.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction

medicinal plants are growing. Due to heavy industrialization,

Ghaziabad is a district ofUttar Pradesh in India,which is one of

the largest industrials area. In the vicinity of industries, many

1828, þ91 9312346815 (moK. Tyagi).2013, JPR Solutions; Publi

plants are bound to absorb industrial polluted water, which

adversely effects their growth, quality and therapeutic values.

After absorbing the polluted water of industries their growth

bile).

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 6 6 7e6 7 3668

becomes stunted and their medicinal value also get reduced.

These plants are binge used as such in medicine and for other

purposes. The manufacturing industries are facing a constant

problem for shortage of genuine and good quality raw mate-

rials. It is therefore essential to ascertain the quality of me-

dicinal plants material before it is employed for the

preparation of drugs. Histo-pharmacognostical study is a key

factor, plays a very important role in determination of

authentication, purity and quality of crude plant drugs or their

parts.

2. Materials and methods

The effluent was analysed by APHA, 1981.1 For anatomical

studies 3rd internode of chenopodium was collected from

both the sites non-polluted (ALTT Centre, Ghaziabad, India) as

well as polluted (Bicycle Industry, Ghaziabad) and studied

according to Metacalf and Chalk, 19502 were consulted; for

chemical analysis Johanson, 1940,3 Youngken, 1951,4 Crom-

well, 19555 & Trease and Evans, 19836 were followed. TLC was

done according to the WHO, Geneva, 1998.7

3. Results

3.1. Effluent analysis

The effluent was analysed and the results are given in Table 1.

3.2. Macromorphology

The plant is an erect or ascending, green or reddish, herb, upto

3.50 m in height. Stem is angular, rarely slender often striped

green red or purple in non-polluted areas, whereas in polluted

areas, stem is purple or red in colour. Leaves in non-polluted

areas are variable in size, shape and dark green in colour.

These are rhomboid, deltoid to lanceolate, upper entire, lower

Table 1 e Comparative account of physico-chemical Character

S. no. Parameters Characteristiceffluents

1. Colour Yellowish

2. Odour e

3. pH 4e6

4. Suspended solids (mg/l) 200 mg/l

5. Total dissolved solids (mg/l) 810 mg/l

6. Total suspended solids (mg/l) 1010 mg/l

7. Dissolved solids (mg/l) 720 mg/l

8. Total solids (mg/l) 840 mg/l

9. BOD (mg/l) 16.0 mg/l

10. COD (mg/l) 200 mg/l

14 Chromium (Cr) 5 mg/l

15. Nickel (Ni) 12 mg/l

16. Zinc (Zn) 15 mg/l

17. Cadmium (Cd) 4 mg/l

18. Copper (Cu) 4 mg/l

19. Temperature 50 �C

toothed or regularly lobed; petioles long slender, often equal

or longer than the blade, petiole is 10e15 cm long; leaf is

1.30e4.00 � 5.00e7.54 cm2. But in case of polluted area the

colour of leaves is yellow green with white patches, petiole is

4e6 cm long and leaf is 1.50e3.50 � 4.00e6.50 cm. The spikes

are less in number in polluted plants samples. The seeds are

1.5 mm in diameter (Fig.1a and b). The main differences are

tabulated in Table 2.

3.3. Microscopical study

The non-polluted stem showed single layer of epidermis

covered by thin cuticle and non glandular trichomes, hypo-

dermis; 4e5 layers of collenchymatous cells, 4e5 layers of

parenchymatous cortex; single layer of endodermis with

casparian strip. Secondary vascular bundles are present in a

ring and remain embedded in the prosenchyma (conjuctive

tissue). Phloem is interxylary. Vascular bundles are conjoint,

collateral, open and endarch. Pith cells are polygonal with

intercellular spaces (Fig. 2a). But in case of polluted stem there

were 5e6 layers of collenchyma, 5e6 layers of parenchyma

whereas ruptured endodermis; phloem and cambium are in

discontinuous manner. Vascular bundles are smaller in size.

Micro and rosette crystals are present in parenchymatous

cells (Fig. 2b). Non-polluted leaf showed single layer of

epidermis bearing glandular and non-glandular trichomes

covered with cuticle. Stomata are anisocytic and anomocytic

present on both the surfaces of leaf and more frequent on

lower surface 1e2 layers of collenchyma in the upper region

and lower region, 4 vascular bundles inmidrib and presence of

micro and rosette crystals of calcium oxalate in parenchy-

matous cells. The stomatal index was found to be 18.12e19.75

on upper surface and 20.00e22.66 on lower surface in non-

polluted leaves while in case of polluted plant samples sto-

matal index is 18.11e23.15 on upper surface and 18.03e22.25

on lower surface. Palisade ratio is lower in polluted leaves.

Vein Islet Number and Vein Termination Number were higher

in those plants which are colleted from polluted areas.

istics of industrial effluent of selected industry.

of Max. recommendedconcentration

Authority/reference

Should be absent I.S.I.: 2490

Odourless I.S.I.: 2490

5.5e9.0 I.S.I.: 2296

e e

2100.0 I.S.I.: 3307

600.0 I.S.I.: 3306

e e

2700.0 e

30.0 I.S.I.: 2490

250.0 I.S.I. 2490,1982

e e

e e

e e

e e

e e

e e

Fig. 1 e Macromorphological differences between Chenopodium album Linn. growing in non-polluted (a) and polluted areas (b).

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 6 6 7e6 7 3 669

Mesophyll is differentiated into 3e4 layers of palisade, 2e3

layers of spongy parenchyma, (Fig. 3a and b). But the polluted

leaf is isobilateral in nature containing 2e3 layers of collen-

chyma present in upper region and 1e3 layers of collenchyma

in lower region. 7e9 layer of palisade with a duct and a

continuous layer of rosette crystals of calcium oxalate Lam-

ina. In polluted leaves the glandular trichomes and spongy

parenchyma are absent (Fig. 3c & d).

Table 2 e Colour reaction tests of Chenopodium album Linn. gro

S. no. Reagents Test

1. Dragenorff’s reagent {Cromwell (1955)} Alkaloid

2. Phloroglucinol þ HCl Lignin

3. FeCl3 Tannin

4. Molisch test Carboh

5. Xanthoproteic test Protein

6. Benedict’s reagent after heating Sugars

7. Sample þ heating with strong KOH þ H2SO4 Subern

8. Molisch test after hydrolysis Glucosi

9. Plant powder þ H2O þ shake Saponin

10. Mg powder þ Conc. HCl Flavin

11. Libermann’s Buchard reagent Steroid

12. Sudan IV Oils

3.4. Preliminary colour reaction tests

The result shows the presence of saponin, tannin, lignin,

protein, carbohydrates, suberin, glucoside, flavin, and traces

amount of oil and absence of alkaloids and sugars in both the

cases. Degrees of changes in colour reaction tests are tabu-

lated in Table 2.

wing in non-polluted and polluted areas.

for Nature of colour Degree of changes

NP Polluted

Negative e e

Dark red þþþ þþBlack þþþ þþ

ydrates Red þþþþ þþYellow þþþþ þþþNegative e e

in Red Black þþþþ þþde Yellow þþþ þþ

Large Froth þþþþ þþGreen - Black þþþþ þþ

s Violet þþþþ þþRed þþþ þþ

}

}

}

Tri.Cu.

Epi.

Col.

Par.

En.S.Ph.

Ca.

X.V.

Pro.In.Ph.

M.Cry.

M.V.B.

Pith

}}

}R.Cry.

a b

Fig. 2 e Anatomical differences in the stem of Chenopodium album Linn. growing in non-polluted (a) and polluted areas (b).

Abbreviations of Figures B.S. e Bundle sheath; Ca. e Cambium; Chl. e Chlorenchyma; Col. e Collenchyma; Cu. e Cuticle;

CV e Coefficient of Variation; D e Diameter; Epi. e Epidermis; En. e Endodermis; F e Frequency; L e Length; M. Cry. e Micro

crystal; M.V.S. e Medullary vascular bundle; Par. e Parenchyma; P.L. e Palisade layer; Peri e Pericycle; Ph. e Phloem; R e

Range; R. Cry. e Rosette crystal; Ra. e Raphides; S. e Stomata; S. Par. e Spongy parenchyma; SD- Standard deviation; Tri. e

Trichome; V.B. e Vascular bundle; X.V e Xylem vessels; W e Width.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 6 6 7e6 7 3670

3.5. TLC

The numbers of spots are higher in non-polluted plant

than the polluted plant (Fig. 4). Rf values of Chenopodium

album Linn. were decreased in those plants which were

collected from polluted areas, results are tabulated in

Table 3.

3.6. Physical evaluation

3.6.1. Extractive values and Ash valuesThe percentage of water and alcoholic soluble extractives are

lower whereas LOD, total ash, acid insoluble and sulphated

ash are higher in polluted plant samples (Table 4).

4. Discussion

The effluent sample was analysed for different physico-

chemical parameters which showed higher values as

compared to the standard values recommended by the Indian

Standard Institute, Similar results were also obtained by

Vijayavathi et al, 2008.8 A critical observation on the data

studied clearly indicate that plants growing at polluted sites

were badly affected and there was a significant reduction in

number of parameters studied as compared to the plants

growing at the control sites. Morphological characters were

found to be decreased in polluted plant samples. Similar ob-

servations were recorded by Angadi and Mathad, 19989 who

have studied the effects of Copper, Cadmium and Mercury on

Tri.Cu.S.Epi.

P.L.B.S.S.Par.

V.B.Col.

M.Cry.R.Cry.

Par.

a

b

0.05mm

b & d

0.01mm

a & c

Cu.S.

Col.Epi.

P.L.

Tri.

V.B.M.Cry.

D.

R.Cry.

Par.

c

d

Fig. 3 e Anatomical differences in the leaf of Chenopodium album Linn. growing in non-polluted (a & b) and polluted areas

(c & d). Abbreviations of Figures B.S. e Bundle sheath; Ca. e Cambium; Chl. e Chlorenchyma; Col. e Collenchyma; Cu. e

Cuticle; CV e Coefficient of Variation; D e Diameter; Epi. e Epidermis; En. e Endodermis; F e Frequency; L e Length; M. Cry. e

Micro crystal; M.V.S. e Medullary vascular bundle; Par. e Parenchyma; P.L. e Palisade layer; Peri e Pericycle; Ph. e Phloem;

R e Range; R. Cry. e Rosette crystal; Ra. e Raphides; S. e Stomata; S. Par. e Spongy parenchyma; SD- Standard deviation; Tri.

e Trichome; V.B. e Vascular bundle; X.V e Xylem vessels; W e Width.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 6 6 7e6 7 3 671

the morphological, physiological and biochemical character-

istics of Scenedesmus quadricauada (Turp) de Breb. and found

maximum inhibition in the growth, chlorophylls, total DNA,

total RNA and protein contents of cells at the sites of higher

metal concentrations. Therefore, it is observed from various

studies that the same species respond differently under

different conditions polluted and non-polluted.

The stem anatomy of polluted plant samples when

compared with those plant samples which were collected

from control sites showed common characteristics viz. both

type of trichomes, collenchymas, parenchyma, pericycle,

medullary vascular bundles open and endarch vascular bun-

dles, but the ruptured endodermis presents only in polluted

plant samples. Reduced secondary growth observed in pre-

sent findings in polluted plant samples goes in conformity

with the result of Jabeen and Abraham, 1998.10 Chaudhari and

Patil, 200111 also observed the inhibition and stimulation in

xylem and phloem in pith region of several plant species

growing under the stress conditions of polluted water. The

reduced length of vessel elements coupled with their

augmented frequency appears to be the significant adapta-

tions to the stress of pollution.

Microscopical studies relatedwith leaf anatomy of polluted

plants samples indicated that less trichomes frequency, less

number of stomata, presences of collenchyma layers, reduced

layer of spongy parenchyma with smaller cell sizes, lesser

ground tissue, decreased ratio of stomatal index and palisade;

more numbers of crystals with bigger size in leaves of polluted

plant samples. Salgare & Acharekar, 199112 have also reported

a considerable decrease in size and frequency of stomata and

epidermal cells of plants growing in polluted environment.

Low stomatal frequency observed in the plants grown in

polluted areas, may reflect adaptation of ecotypic significance

in regulating the limited and controlled entry of harmful

gaseous pollutants into the plants tissues, especiallywhen the

plant grown in polluted area. The response of plants varies in

accordance to varying nature of pollutants their concentra-

tions. Powder analysis of Chenopodium showed that elements

of xylem and phloem were smaller in size in polluted plant

samples. Although the pollution effect is very prominent in

Fig. 4 e Number of spots of Chenopodium album Linn.

growing in non-polluted (a) and polluted areas (b).

Table 3 e The Rf values of Chenopodium album Linn.growing in non-polluted and polluted areas.

S. no. Wavelengths Non-polluted Polluted

Rf values Rf values

1. Sunlight (visible) 0.30, 0.42,

0.85, 0.89

0.42, 0.89

2. U.V. light (264 nm) 0.30, 0.34, 0.42,

0.85, 0.89

0.34, 0.36,

0.42, 0.89

3. U.V. light (365 nm) 0.30, 0.42,

0.85, 0.89

0.34, 0.42, 0.89

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 6 6 7e6 7 3672

several aspects of growth and development of the plant, it

significantly promotes the number of vessels and fibres in

plants growing under pollution effect.

Physico-chemical of powdered drug evaluation includes

fluorescence behaviour, extractive and total ash values. The

polluted plant samples showed quick differentiations to

fluorescence behaviour. Water and alcohol extractive values

were found to be lowered collected from polluted areas. Ash

values were comparatively higher in polluted plant samples.

Similar observations weremade by Sharma and Habib, 1995.13

Table 4 e Extractive values (%) and Ash values (%) of Chenopodiu

Extractive values(%) and Ash values(%)

S. no. Parameters N

1. Water Soluble 24.887 �2. Alcohol Soluble 36.825 �3. LOD 22.509 �4. Total Ash Value 12.910 �5. Acid Insoluble 2.271 �6. Sulphated Ash 28.920 �

Significant at 0.1% e * 1.0% e ** 5.0% e ***.

Percentage of ash content was higher in the plant samples

those collected frompolluted areas as compared to the control

one, because ash content of plants is the direct manifestation

of bio-accumulation of minerals absorbed as macro and

micronutrients which take up different functions. The per-

centages of extractive values were lower and ash values were

higher in polluted plants. From the observations some alter-

ation in the bio-chemical parameters were recorded in the

plants growing near the industrial effluent. The amount of

chemical constituents found to have decreased in those

plants which were growing in polluted areas.

From the observations of TLC, it was seen that the number

of spots were decreased in the plant samples of polluted sites.

From the findings of this investigation it may be safely

asserted that there had been qualitative and quantitative al-

ternations in the chemical constituents in the plants growing

in industrial areas (polluted). It would not be unwise to state

that industrial pollution might have also lowered the drug

potency of the plants growing in the vicinity of industries.

Almost similar observations were recorded by Dhar et al,

2003.14

In order to determine the quality of medicinal plants with

regard to its authenticity histo-pharmacognostical characters

viz. macroscopical, anatomical, chemical analysis, TLC,

extractive values and ash values are very important. Anatomy

often proves very useful for individual identification of plants

so microscopical methods are of great value towards their

identification and authentication of the authenticity of plant

drugs. They provide evidences concerning relationship of

groups such as families or help to establish affinities of genera

of uncertain taxonomic status. The number of stomata and

epidermal cells, vein-islets and vein termination number per

unit area, palisade ratio, stomatal index etc. give constant

m album Linn. growing in non-polluted and polluted areas.

on-polluted Polluted

0.561; CV ¼ 2.254 21.827 � 0.381***; CV ¼ 1.745

0.683; CV ¼ 1.854 32.620 � 0.741***; CV ¼ 2.271

0.910; CV ¼ 4.043 24.887 � 0.830; CV ¼ 3.335

1.100; CV ¼ 8.520 18.650 � 1.125***; CV ¼ 6.032

0.160; CV ¼ 7.048 4.547 � 0.430*; CV ¼ 9.456

0.531; CV ¼ 18.360 38.000 � 0.510*; CV ¼ 13.421

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 6 6 7e6 7 3 673

structure for different species of plants. Moreover, different

types of stomata, crystals, fibers, trichomes etc. present in

powdered drug help in the identification of plants or differ-

entiation in comparison of same plant species, which are

collected from the industrial and non-industrial localities.

5. Conclusion

However we may conclude that the plants from non-polluted

area should be collected for quality production of medicines,

since majority of parameters reflect decreasing data values in

the plants taken from polluted area.

Conflicts of interest

All authors have none to declare.

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