Received: 26 November 2019 Revised: 14 December 2019 Accepted: 18 December 2019

DOI: 10.1002/jemt.23430

RESEARCH ARTICLE

Anatomy, micromorphology, and physiochemical analysis of

Rhus succedanea var. himalaica root

Shafqat Ali Khan1 | Barkatullah1 | Behramand Khan2

1
Department of Botany, Islamia College,
Peshawar, Pakistan
2
Department of Chemistry, Islamia College,
Peshawar, Pakistan
Correspondence
Shafqat Ali Khan, Department of Botany,
Islamia College, Peshawar, Pakistan.
Email: shafqatbotany3@gmail.com
Review Editor: Paul Verkade
Abstract
Root micromorphology can play a vital role in the systematics of angiosperms to
understand the complexity among different genera and species. Present study is
about microscopic and physiochemical evaluation of Rhus succedanea root belonging
to the family Anacardiaceae. Various organoleptic characteristics of root were stud-
ied. Microscopy showed that transverse section of the root appeared rounded and
presented a typical histological differentiation having different average cells length
and width. Under light microscopy and scanning electron microscopy, the powder
revealed the existence of pitted xylem vessels, phloem sieve elements, phloem fibers
and cork cells, and so forth. Fluorescence study of the powder showed various
shades of color that gives a valuable information regarding characterization, authenti-
cation, and identification of the plant material. These documented information can
be acted as record and monograph of a specific plant materials. Nutritional composi-
tion of root showed that Ash, fat, protein, carbohydrates, and total gross energy were
higher in summer as compared to winter season. On the other hand, moisture and
fibers were higher in winter and declined in summer. Root powder gave highest
extractive value (37.3%) in methanol and showed the presence of various groups of
secondary metabolites qualitatively while, quantitatively flavonoids (0.18 mg/g) was
detected in highest amount. The above parameters, being reported for the first time
and are significant toward establishing the microscopic and pharmacognostic stan-
dards for future identification and authentication of genuine herbal drug. Root micro-
morphology can be used as an additional tool to aid description and to distinguish
many complex taxa and that is of significant value for the taxonomic assessment of
this genera and species.

KEYWORDS

extractive values, fluorescence analysis, microscopic study, phytochemical screening,

proximate analysis, Rhus succedanea, root

1 | I N T RO DU CT I O N causing skin irritation and severe dermatitis to local sensitive and

Rhus succedanea var. himalaica (Figure 1) is a perennial medium size
deciduous plant that grows from 5 to 9 m in length, locally called as
Rakhkal in Pashto and Kakarsingi or Arkhol in Urdu. The plant release
white latex on cut and injury which is thought-out to be allergic,
allergic inhabitants, whenever the body of a person comes in contact
with the plant or its latex (Hiraoka & Kuramoto, 2004). In Pakistan,
the plant is distributed in innermost ranges of the Himalaya from
1,524 to 2,450 m in Shangla, Kashmir, Swat, Dir. Rhus (lacquer)
chemically composed of volatile oil, phenols, glycoproteins, gummy

Microsc Res Tech. 2020;1–12. wileyonlinelibrary.com/journal/jemt © 2020 Wiley Periodicals, Inc. 1

2 KHAN ET AL.
mucilaginous substance, and enzyme laccase (Johnson & Johnson,
2006). Previous literature shows that root of R. succedanea has tonic,
expectorant, antiviral, and blood clotting properties. The fruit and pulp
is also used in candies, wax matches, pencils, as substituent of bees'
wax, in softening of pomades in lipsticks, textile, furniture, leather pol-
ishes, and also used as Ayurvedic remedy for diarrhea and dysentery
(Antonisamy, Aparna, Jeeva, Sukumaran, & Anantham, 2012).
The pharmacognostic and botanical characterization and descrip-
tion of a plant is necessary to get the desired and genuine drug for
good medicinal effect. The main aim of pharmacognosist is to evaluate
the importance of natural crude materials from various parts of plant
to the required standard by passing through strict standard proce-
dures of pharmacognosy. That reduces the wrong recommendations
of medicinal plants and traditional medicines to high extent (Ickert-
Bond, 2000). Although many other visible, macromorphological and
organoleptic features are commonly used for description and explana-
tion of species in floras. The use of root micromorphology and anat-
omy can be an additional and distinguishing tool for correct
description and differentiation between many complex taxa and have
of substantial value for the taxonomic assessment of the genus (Khan
et al., 2018). The root micromorphology has wide taxonomic and
paleobotanical implications role in the systematics of angiosperms to
know the complexity that exists in the macromorphological characters
among the different classes, families, genera, and species (Mickle,
Lumaga, Moretti, & De-Luca, 2011). The present study aimed to
investigate different pharmacognostic and micro-morphological char-
acters of R. succedanea root using light and scanning electron micros-
copy (LM and SEM) to find how these features are useful for
standardization of crude drugs and identification of different genera
and species of angiosperms.
F I G U R E 1 Tree of R. succedanea var.
himalaica J. D. Hook [Color figure can be
viewed at wileyonlinelibrary.com]
2 | METHODS
2.1 | Collection and preservation
The fresh roots of Rhus succedanea var. himalaica were collected from
Shalmanay, Kotkay, District Shangla located at 34a
310 to 33
080
North latitude about 72
330 to 73
010 East longitude and elevation of
the area is about 3,000 m above the sea level and area characterized
by moist temperate climate. Roots were collected by digging in large
quantity, cleaned, washed, separated, cut into various size fragments
and were shade dried for 2 weeks. The roots were ground by electrical
grinder, passed through fine sieve#30 size 600 μm and 500 g was used
for microscopic, nutritional, and elemental analysis. The remaining
powder was kept in sterilized sealed bottles to keep away from pests,
pathogens, humidity, and harsh conditions and made in to extract
which were later used for phytochemical analysis.
2.2 | Morphological observation
The morphological and organoleptic features (include shape, color,
size, odor, taste, etc.) of root of R. succedanea were screened out
organoleptically following method of Wallis (1985).
2.3 | Anatomical study
The anatomical study of root of R. succedanea was done in Pharma-
cognosy Lab, Islamia College, by cutting thin sections of each with the
help of sharp razor by mounting in fresh potato tuber slices. The
KHAN ET AL. 3

thinnest section of each part was selected, stained for 2–3 min in saf- 2.7 | Pharmacognostic parameters
ranin and dehydrated with alcoholic series of different grades (10, 20,
30, 40, 50, 60, 70, 80, and 90%). Finally, Canada balsam mounted on Other pharmacognostic studies like extractive values determination,
slides with the help of dropper to make permanent slides and the fluorescence analysis, and qualitative and quantitative determination
slides were fixed under the microscope. Clear images of Transverse was done following the standard methods of Hegde, Jayaraj, and
Section were taken by connecting the Labomid digital Microscope Bhandarkar (2015); Evans (2009); and Harborne (1998), respectively,
model LB-212 features a built in 1.3 Megapixel Camera with com- with slight modifications.
puter system. The average size of cells (length and width) in various
tissues was determined by micrometry with the help of stage and ocu-
lar micrometer. 3 | RESULTS AND DISCUSSION

3.1 | Macroscopy and organoleptic characteristics

2.4 | Investigation of powder drug through LM
Assessment of the powdered drug was done in Pharmacognosy Lab,
Department of Botany, Islamia College Peshawar Pakistan, by method of
Evans (2009). Dried roots were ground by electrical grinder, passed
through fine sieve#30 size 600 μm and were mixed with in concentrated
chloral hydrate and boiled for 15 min. Small drops of solution containing
powder were put on clean slide, macerated in glycerin and iodine solu-
tion, and observed under Labomid digital compound Microscope model
LB-212 features a built in 1.3 Megapixel Camera with computer system
microscope for various all structures and photographs were taken.
2.5 | Scanning electron microscopy
The methodology of Terrel and Wergin (1979) with slight modification
was used for SEM analysis. The dried fine powder of root passed
through sieve#30 size 600 μm were mounted on gold-coated stubs
through double-coated scotch tape exposing one side. JFOL-JFC, 1100E
ion sputtering device was used to sputter the specimens with gold–palla-
dium. These specimens were then observed using SEM (Model JSOL
JEM-5910) installed in the Central Resource Library (CRL), Department
of Physics University of Peshawar. Photographs of the specimens were
taken using Polaroid P/N666 film and the average length and width of
various cells were measured using standard scale procedure.
Above the soil visible, macromorphological and organoleptic features are
commonly used for description and explanation of species in floras root
micromorphology and anatomy was not considered by taxonomist as a
valuable descriptive tool, but according to many taxonomist its can play
a significant and crucial role in description and resolving many complexi-
ties in closely related taxa, genera and species. The present study was
conducted in such regards including macroscopical and organoleptic
investigation of the fresh and dried root of Rhus succedanea. var.
himalaica. Organoleptic evaluation showed that the average length and
breadth of fresh collected root fragments was 13–20 cm and 1.5–4 cm,
while of dried was 11–18 cm and 1.3–3.8 cm, respectively. Color was
brown to brownish yellow, cylindrical in shape and rough in appearance,
odor was aromatic in fresh form and become indistinct when dried, tex-
ture was flexible when fresh and become hard and tough on drying with
uneven fibrous texture surface and astringent taste (Table 1).
3.2 | Microscopy and histology
In Transverse section, the root of R. succedanea appeared rounded
and presented the following tissues. Measurement of cells in the
T A B L E 1 Morphological and organoleptic evaluation of R.
succedanea var. himalaica root

Features Fresh Dry

Size Length = 13–20 cm; Length = 11–18 cm;
2.6 | Proximate nutrient determination
width = 1.5–4 cm width = 1.3–3.5 cm
Color Yellowish to dark brown Yellowish brown to
Moisture contents, minerals ash, crude protein, fat, and insoluble
creamy
fibers were determined by standard methods of AOAC (2005). Total
Shape Cylindrical Cylindrical
% carbohydrate was calculated by formula of Onyeike, Olungwe, and
Odor Agreeable Indistinct
Uwakwe (1995) as
Direction of Vertical downward Vertical downward
growth
Total%carbohydrate = 100−%crude protein + %fiber + %fat
Fracture Flexible Hard
+ %moisture + %ash
Texture Rough Rough
Texture Uneven and fibrous Uneven and fibrous
The caloric value in (kcal) (total gross energy) was estimated by
surface
using the following formula. Energy value (kcal/100 g) = (2.62 × %
Taste Astringent Indistinct
protein) + (8.37 × %fat) + (4.2 × %carbohydrate). Each determination
Smell Acceptable, pleasant Pleasant
was repeated three times and was recorded as mean ± SEM.
 4 KHAN ET AL.

T.S. was taken in triplicates and represented as Mean ± SEM (standard

27 ± 2.11

17 ± 1.09

10 ± 0.22
error of mean) (Table 2).
Width

Width

Width
Cork: It is the outermost boundary composed of two to four layers

Phloem fibers

Phloem fibers
31 ± 4.78 of cells, compactly arranged and dark brown in color. The cells of cork

40 ± 3.55

60 ± 3.88
were cylindrical in structure and surrounded by thick walls. The aver-
Length

Length

Length
age length and width of cork cells were 17 and 19 μm, respectively.
Pith

Phellogen: Below the cork, there is a single layer of phellogen, com-

30 ± 1.98

7 ± 0.012 posed of thin walled spherical isodiametric cells with average length

5 ± 0.21
Width

Width

Width
and width 12 and 9 μm, respectively. Cortex: The phellogen is followed
by a multilayered cortex composed of irregular polygonal collenchyma
Xylem fibers

Xylem fibers

and parenchyma cells. It occupies the largest area in root with average
41 ± 1.85

45 ± 2.77

55 ± 3.99
Length

Length

Length
cell length of 29 μm and width of 14 μm. Latex ducts or canals: The
Xylem

multilayered cortex is interrupted by latex canals or oil cavities that

contain white latex which oozes out on injury. The latex canal having
21 ± 1.22

16 ± 1.54

17 ± 2.99

30 μm length and 23 μm width. Vascular bundles: Inner to the cortex is

Width

Width

Width
Fragments of Cork

Fragments of cork

a ring of collateral vascular bundles in which the phloem was toward

the outer side while xylem was in the direction of central pith region.
33 ± 3.78

25 ± 2.22

22 ± 1.33

The phloem visibly exhibited the phloem fibers, phloem parenchyma,

Phloem
Length

Length

Length

and sieve tubes. The xylem was toward the center, occupied large area
and composed of radially arranged xylem vessels occupying the entire
Irregular calcium oxalate

Irregular calcium oxalate

central portion, interconnected through collenchyma cells and medul-

14 ± 1.99

60 ± 2.99
6 ± 0.12
Width

Width

Width

lary rays. Also showed the presence of vertically arranged xylem fibers.
In between the vascular bundles, the cambium was located that is
Microscopical measurements (μm) of various tissues of different of R. succedanea var. himalaica root

responsible for secondary growth in roots. The average length and

29 ± 4.22

80 ± 3.99
crystal

width of phloem sieve cells and xylem vessels were (33 and 21 μm)
8 ± 0.22
Length

Length

Length
Cortex

and (41 and 30 μm), respectively. Center of the root was occupied by
small parenchymatous pith, composed of irregular-, polygonal-, and
oval-shaped cells, with thin walls with average cell length of 31 μm
Prismatic calcium oxalate
23 ± 2.59

28 ± 1.44
9 ± 1.22

and width of 27 μm (Figures 2 and 3 and Table 2).

Width

Width

Width

The following investigators carried out similar histological work on

roots of various plants and reported similar histological composition.
Starch grains
Latex canal

30 ± 3.09

12 ± 0.54

75 ± 4.11

Soni, Gupta, Solanki, and Jana (2011) documented that the root of
crystal
Length

Length

Length

Pavonia odorata showed similar histological composition like cork, cortex

having parenchymatous, pericycle, collateral vascular bundles, and cen-
tral pith. Xavier, Devkar, Chaudhary, Shreedhara, and Setty (2015)
19 ± 1.22

10 ± 1.98

Abbreviations: LM, light microscopy; SEM, scanning electron microscopy.

9 ± 1.67
Width

Width

Width
Sieve elements

Sieve elements
12 ± 0.987
Phellogen

40 ± 0.33

29 ± 4.55
Length

Length

Length
19 ± 2.45

17 ± 1.55

11 ± 1.77
Pitted xylem vessels
Width

Width

Width
Xylem vessels
17 ± 1.23

34 ± 3.09

27 ± 2.55
Length

Length

Length
Cork
Transverse section

Powder SEM
TABLE 2

Powder

F I G U R E 2 Transverse section of R. succedanea var. himalaica J. D.

LM

Hook. root [Color figure can be viewed at wileyonlinelibrary.com]

KHAN ET AL.
F I G U R E 3 Average length and width
in (μm) of various cells in T.S. of R.
succedanea var. himalaica root [Color
figure can be viewed at
wileyonlinelibrary.com]
carried out LM of root Malva parviflora, Gupta and Rao (2012) worked
on root of Fumaria indica (Hausskn and reported that root was almost
circular in outline and consisted of epiblema, endodermis, collateral type
vascular bundles, and central pith. Various other workers have also been
documented the same anatomy of roots of Nothosaerva brachiate,
Bergenia ciliata, and Homonoia riparia (Ghimare, Ghimare, & Heo, 2012;
Madhavan, Goswami, Gurudeva, & Yoganarasimhan, 2010 and Nassar &
Ortiz, 2007). Our understanding is in line with Eshel and Beeckman
(2013) who reported that roots, the “hidden half” of plant, is even more
fundamental than aerial parts shoots, leaves, and flowers. Root performs
much biological functions like anchorage in soil, storage of important
contents, resources acquisition tool, and communication with other
plants organism and environment. Plant breeders, taxonomists, and den-
drologists very rarely considered the roots because it is very difficult to
observe and study in situ (Stein & Nothnagel, 1995). There is a strong
relationship between anatomical basis with morphology, development,
strength, habitat, and physiology of plant as they are dependent on the
proportion of different types of tissues in the root. Slow growing and
very tall plants species possess very deep and dense roots system that
increases resistivity and longevity of plant (Thomas, Graham, & Hayden,
2016). The root micromorphology has wide taxonomic and paleobotani-
cal implications and is considered more vigorous and authentic than flo-
ral and palynological studies. Root micromorphology can play a dynamic
role in the systematics and classification of angiosperms to understand
the complications that exists in visible above ground physical and mor-
phological features among the closely related different classes, families,
genera, and species (Mickle et al., 2011). In conclusion, the structures,
histology, and micrometry of root of R. succedanea enhanced our under-
standing of the biological features of the family Anacardiaceae and its
adaptation to specific habitat. It also provides a clue and a valuable taxo-
nomic tool for differentiation among species of the same genera.
3.3 | Powder LM and SEM evaluations
Powder drugs of the root was white creamy in color, astringent in
taste, with indistinct odor and soft in texture. Various cells and tissue
5
were observed and their measurement was taken as Mean ± SEM in
Table 2. Under LM and SEM, the powder revealed the existence of
pitted xylem vessels having average length and width of 34 and
17 μm (LM), 27 and 11 μm (SEM), respectively. Phloem sieve elements
with length of 40 μm and width of 19 μm (LM) and 29 μm length and
10 μm width (SEM) were recorded. Phloem fibers and cork cells with
average length and width of 25 and 16 μm (LM), 22 and 17 μm (SEM)
were also observed (Figures 4–6 and Table 2). Starch grains and root
hairs were detected only in LM while, prismatic crystals were found in
only SEM. Many other tissues like, xylem tracheids, fibers, pith cells,
and fragments of cortex parenchyma were also observed, their aver-
age length and width were given in (Figures 4–6 and Table 2).
Various investigators reported similar cells and tissues while
studying powder of different parts of several medicinal plants.
Powder microscopy helps in both the identification of the herbal
drugs and detection of adulteration in crude drugs (Soni et al., 2011).
Similarly, Pandavadra and Chanda (2014); Xavier et al. (2015); Samanta,
Yadav, Tripathi, Kumar, and Hossain (2013); and Nassar & Ortiz, 2007
reported same type of tissues in roots of Odina wodier, Limonia
acidissima L. Homonoia riparia, Psidium guajava, Coccinia indic, Memecylon
umbellatum, Crotalaria juncea L., and Calotropis procera, respectively.
These works strongly support the present study. It is an easy step to dis-
criminate adulterated drug from the unadulterated ones.
3.4 | Proximate nutrient determination
The proximate nutritional analysis of the root was done in both sum-
mer and winter seasons. The data were represented in Table 3 and
Figure 7. Plants are important source of various kinds of nutrients
such as carbohydrates, ash, moisture content, fats, protein, vitamins,
and so forth and play an important role in human, animal, and plants
life. These nutrients are not only necessary for shielding health of liv-
ing organisms from various diseases but also help in maintains of
health in proper condition from adverse effects of environment
(Kokate, 2008; (Atasie, Akinhanmi, & Ojiodu, 2016). Carbohydrates,
fats, and proteins are sources of energy in diet and provide raw
6 KHAN ET AL.

F I G U R E 4 Light microscopy (LM) of R. succedanea var. himalaica root powder. (a) Pitted xylem vessels, (b) phloem fibers, (c) bundles of
phloem fibers, (d) fitted Xylem vessels and starch grain, (e) single xylem vessel and calcium oxalate crystal, (f) cork cells, (g) fragments of cortical
cells, (h) xylem fibers, (i) phloem fiber, (j) root hair, (k) xylem sieve elements, and (l) pith cells [Color figure can be viewed at wileyonlinelibrary.com]

materials to various industries for different products development.
Moisture provides medium for various metabolic activities and low
percent moisture contents in root would prevent and hinder the
reproductive growth and spread of microorganisms and extend shelf
life of powder drugs and food products (Vogel-Mikuš, Pelicon,
Vavpetic, Kreft, & Regvar, 2009). The ash is the amount of inorganic
salts and minerals naturally present or preserved in the powder drug
and provides basis for finding the cleanliness of a drug and gives infor-
mation about the adulteration with inorganic matter in the form of
sand, earth, or lime (Gupta & Rao, 2012). Highest amount of ash in the
root powder suggests a high deposition of minerals elements. Ash
standards have been established for a number of drugs, above or
below of which are not permissible for human administration, that is,
in Tinospora cordifolia 3–16%, in Zingiber officinale 1.7–6%, and in
Rheum rhabarbarum 8–40% is permissible.
In current study, various nutrients, their amount, and percentage
on dry bases in winter and summer seasons were studied. Results are
shown in (Table 3 and Figure 7). Root of R. succedanea showed minimal
fat content. High fat contents consumptions are said to be causes heart
diseases such as atherosclerosis, aging, obesity, and cancer. Dietary fats
increase of palatability of food by absorbing and retaining flavors
(Badugu, 2012). Sufficient amount of dietary fibers in food intake can
reduces the blood cholesterols, risk of coronary heart problems, hyper-
tension, diarrhea, constipation, diabetes, breast, and intestine cancers.
KHAN ET AL. 7

F I G U R E 5 Scanning electron microscopy (SEM) of R. succedanea var. himalaica root powder. (a) Phloem fibers, (b) cork cells, (c) pith cells,
(d) xylem fibers, (e) irregular calcium oxalate crystal, (f) fragment of cortex, (g,h) fitted xylem vessels, (i) phloem fiber, (j) cortical cells, (k) single
xylem sieve elements, and (L) prismatic calcium oxalate crystal [Color figure can be viewed at wileyonlinelibrary.com]

Protein contents of root is in agreement with the research of Juliet,
Jothi, and Rajakumar (2012) that protein level of mostly plants ranged
from 19.58 to 41.46% on dry weight bases. Plant crude food products
that give more than 10% of energy caloric values from considered a
good source of proteins. Nutrients value and energy of medicinal plant
sample are mostly used to interpret medicinal sample consumption as
consumption of food components (Selvam, 2015). The mean gross
energy of the root was highest (219.49 kcal/100 g) in summer and low
in winter (214.711.9 kcal/100 g) (Table 3). These results are supported
by Samanta et al. (2013) and Zunjar, Mammen, Trivedi, and Daniel
(2011) who reported the high gross energy in the roots of Sida
rhombifolia and Carica papaya.
Nutritional composition of root showed that ash, fat, protein, car-
bohydrates, and total gross energy was higher in summer season as
compared to winter season. On the other hand, moisture and fibers
were higher in winter and declined in summer season (Table 3 and
Figure 7). These results showed that with the change of season, the
nutritional composition was affected by the environment. Temperature
and humidity are some environmental factors responsible for the alter-
ation of fiber and moisture contents in plants (Deore, Jajoo, Chittam, &
8 KHAN ET AL.

F I G U R E 6 Average length and width in (μm) of various cells in light microscopy (LM) and scanning electron microscopy (SEM) of powder
of root [Color figure can be viewed at wileyonlinelibrary.com]

TABLE 3 Proximate nutritional analysis of R. succedanea var. himalaica root in different seasons

Gross energy
Moisture (%) Ash (%) Fats (%) Fibers (%) Proteins (%) Carbohydrates (%) (kcal/100 g)
Seasons mean ± SEM mean ± SEM mean ± SEM mean ± SEM mean ± SEM mean ± SEM mean ± SEM
Summer 7.2 ± 0.19 2.39 ± 0.01 0.77 ± 0.001 38.24 ± 3.57 6.2 ± 2.16 46.17 ± 4.091 219.49 ± 1.5
Winter 9.2 ± 3.11 1.39 ± 0.01 0.69 ± 0.001 41.24 ± 3.57 4.2 ± 2.16 40.17 ± 4.091 214.711 ± 1.344

F I G U R E 7 Proximate analysis of
R. succedanea root [Color figure can
be viewed at wileyonlinelibrary.com]

Deshmukh, 2015). Drought is also one of the abiotic factors that
decrease the crude proteins in plants. The outcome of proximate evalu-
ation indicates that R. succedanea root may be used to create alternative
and substituent source of chief dietary component.
3.5 | Fluorescence analysis
The root powder drug of R. succedanea was studied under visible
and ultra violet, short (254 nm) and long wavelength (366 nm) light
for fluorescence characters. The sample was treated with different
reagents that exhibited several colors variations from red, brown,
black, blue, green, pink to dark black, yellowish brown, bluish green,
and so forth with each different chemical reagents, which was
the sign of the existence of different fluorescent biocompounds
and substances (Table 4). Fluorescence investigations gives a
valuable information regarding characterization, authentication and
identification and of the plant material. These documented informa-
tion can be act as a reference and record for future exact identifica-
tion of a specific plant, materials and will also be useful in making a
monograph of the plant. In addition to this, it is a tool for detection
substituents, adulterants and will help in maintaining the quality,
purity reproducibility, and efficacy of natural drug. It is an easy
step to discriminate adulterated drugs from the unadulterated ones.
Many investigators carried worked same research using different
medicinal plants, their different parts and observed same type of colors
KHAN ET AL. 9

T A B L E 4 Fluorescence analysis of
S.no Drugs reagents Visible light UV low (250–270 nm) UV high (360–390 nm)
powdered of R. succedanea var.
himalaica root 1 Drug without reagent Cream Yellow Yellowish brown
2 50% HNO3 Yellow Brown Black
3 Picric acids Gray Dark brown Dark black
4 50% NH3 Brown Black Dark green
5 50% HCl Black Green Dark green
6 H2SO4 Brown Dark brown Dark black
7 NaOH Yellow Pink Brown
8 Iodine Blue Dark blue Black
9 Fecl3 Brown Dark brown Black
10 Methanol Creamy Brown Pink
11 Diethyl ether Creamy Gray Dark brown

F I G U R E 8 Percent extractive values

powder of R. succedanea root in various
solvents [Color figure can be viewed at
wileyonlinelibrary.com]

variations. Chand et al. (2012) and Biswal et al. (2011) reported that
fluorescence is an essential tool to detect all ingredients in powders on
reaction with various chemicals and solvents under ultraviolet (UV) and
infrared lights. They suggested that these can be a indicative tool for
detection of adulteration, substitutions if any. Wallis, 1985 documented
that the UV light is very active in generating fluorescent illumination in
specific chemical compounds that do not show illumination in visible
light so for this purpose UV analysis can be used for determination of
adulteration in crude powder drugs. Some crude powder drugs are
often evaluated qualitatively through this technique (Mbwambo, Moshi,
Masimba, Kapingu, & Nondo, 2009) The fluorescent color is definite for
every chemical substance and is an adequate sensitive procedure and
enables the precise and accurate determination of pharmaceutical sam-
ples. Crude drugs have the quality to show various colors under the
passing of UV light. It is a step toward the authentication and purifica-
tion of crude drugs from impurities.
3.6 | Determination of solvent extractive values
In the present study, the extractive value of root was determined
(Figure 8). The percent extractive values showed that the root
powder gives highest value in ethanol (37.3%) followed by chloro-
form (27.4%), n-butane (25.1%), methanol (24.8%), and distilled
water (23.2%), while it gives the lowest percent yield in ace-
tone (19.2%).
The extractive values confirmed that root powder gives highest
extraction in methanol, so the powder was extracted in ethanol for
further studies in the present work.
Extractive values determine different types of active
phytoconstituents and their amount in medicinal plants on the bases
of nature of constituents and the solvent used. It gives different
amount and types of phytoconstituents with a particular solvent and
used for the detection of adulterants and exhausted materials in
crude drug (Atasie et al., 2016). Extractive solvent separates the
therapeutic and active portion from crude drugs and powder of dif-
ferent parts of plant and is an important indication and determina-
tion of nature of chemical constituents in drugs (Khan & Khan,
2013). Several investigators reported crude powder extractive values
of different plants using a number of organic and inorganic solvents,
which strongly support the significance of this parameter in pharma-
cognostic evaluation, as, for example, Chand et al. (2012); (Vogel-
Mikuš et al., 2009); Khan and Khan (2013), Zunjar et al. (2011), and
Hussain, Fareed, and Ali (2011) investigated the extractive values of
10 KHAN ET AL.

Rosmarinus officinalis L., Spondias pinnata, Moringa oleifera., Alstonia 3.7 | Qualitative and quantitative phytochemical
scholaris., Carica papaya, and Hygrophila auriculata K., respectively. evaluation
These workers concluded and suggested that extractive value deter-
mination is the main and cheap source of detection of adulterants, Root powder was extracted with methanol; the solvent was evapo-
exhausted materials, and selection of suitable solvent for extraction rated through rotary vacuum evaporator. The root extracts showed
of crude powder in which it give highest number of soluble constitu- strongly the presence of carbohydrates, protein in high amount,
ents. In the current research, root gives the highest extractive values amino acids, alkaloids, phenols, flavonoids, terpenoids, and anthraqui-
in methanol. Hence, for further study, the powder was extracted nones, while saponin was just detected. The fixed oil and tannins
with methanol. The present finding will be helpful for future phyto- were not detected at all (Table 5). The amount and percent quantita-
chemical research on this plant. tive phytochemical analysis of ethanolic crude extract of root for alka-
loids, flavonoids and sterol showed that high number of flavonoids
(0.18 mg/g), which amount to 18% was found in roots followed by
alkaloids (0.15 mg/g) 15% while sterols was detected to be low, by
T A B L E 5 Qualitative screening tests of R. succedanea var.
himalaica root 11% only (Figure 9.).
The therapeutic implication of natural plants is mainly dependent
S.no Constituents Chemical tests Root
on the presence of active secondary phytoconstituents like flavo-
1. Carbohydrates Benedict test ++ noids, alkaloids, anthraquinone, terpenoids, and phenolic compounds.
Fehling test ++ Qualitative and quantitative phytochemical screening must be respon-
2 Protein and amino acids Ninhydrin test ++ sible for the detection of secondary metabolites in plants crude mate-
Biuret test ++ rials, having the pharmacological amplifications of the crude drugs and
Xanthoproteic test + provides genuine drugs for companies and public health (Rai, Pai,
3 Alkaloids Mayer's test ++ Kedilaya, & Hegde, 2013). Flavonoids showed to possess the ability of

Wagner's test ++ altering immunological response and also have anti-anaphylactic, anti-
inflammatory, antioxidant, anti-allergic, antimicrobial, and anticancer
4 Phenols FeCl3 test ++
effects (Yun, Lee, Kwon, & Choi, 1996). Flavonoids have been
5 Flavonoids Alkaline reagent test +
reported to be used as antioxidant, analgesic, and free-radical scaven-
Lead acetate test ++
ger, and prevent the menopausal symptom in female (Antonisamy
6 Fixed oil −
et al., 2012). The astringent properties of plants suggested being due
7 Saponins Frothing test +
to the presence of high amount of steroid and terpenoids, saponins
Foaming test + and shows relationships with sex hormone and possessing strong
HCl test − analgesic effect. The tannins are used in bacterial, viral infections,
8 Terpenoids Salkowski's test ++ burns, inflammation, and wound healing (Savithramma, Rao, &
Copper acetate test + Suhrulatha, 2011). The saponins and glycosides have to be used
9 Tannins FeCl3 test − as immune-regulatory, anticancerous and in most of the cardiac dis-
Alkaline reagent test − eases. Phenolic phytoconstituents documented to show toxicity
10 Anthraquinones Borntrager's test +++ against pathogens, like bacteria and shows cytotoxic, anti-mutagenic,
anti-oxidative, and astringent properties (Edeoga & Eriata, 2009).
Note. −, not detected; +, detected; ++, strongly detected.
Anthraquinone metabolites are used as laxative, antimalarial, and anti-
neoplastic (Deore et al., 2015).
Similar work was carried out by various investigators, for example,
Yakubu & Salimon, 2015 carried out the phytochemicals investigation
of root of Mangifera indica L. (Anacardiaceae). Al-Snafi (2015); Shweta,
Ganesh, and Somshekhar (2012); Shilpashree, Dang, and Das (2015);
and Soni and Sosa (2013) worked on different extracts of various
plants viz, Chenopodium album, Alectra parasitica, Pueraria tuberosa,
Ipomoea mauritiana J., Adenia hondala, and Cycas circinalis for detec-
tion of phytoconstituents both quantitatively and qualitatively. Cor-
rect identification, authentication and quality assurance of the
preliminary resources is an important requirement to make sure the
reproducible quality of phytomedicine which will show the safety
and effectiveness of herbal products (Shweta et al., 2012). Pharma-
F I G U R E 9 Quantitative phytochemicals of R. succedanea root cognosy is an easy step to discriminate adulterated drug from the
[Color figure can be viewed at wileyonlinelibrary.com] unadulterated ones.
KHAN ET AL.
All these reports suggest the importance of qualitative and quan-
titative phytochemical screening of crude drugs, which greatly helps
the researchers in the field of photochemistry and pharmacology to
work advance research on medicinal plants in their respective field.
The information obtained from the present pharmacognostical studies
will be used for supplementary pharmacological and therapeutical
evaluation, correct identification, verification, authentication, and
quality assurance of the preliminary resources. The presence and
quantity of the phytochemicals shows the safety and effectiveness of
herbal products. The present work on root of R. succedanea will be
provide a basic step and great help to the researchers for further
advance and deep work on isolation, identification, characterization
and specific effect of phytochemicals detected and can be used as a
cheap and safe drugs and medicine.
4 | C O N CL U S I O N A ND
RECOMMENDATION
The current Microscopic and pharmacognostic study of the root of
R. succedanea is done for the first time. These findings encourage, tax-
onomists and pharmacognosists to use morphological and anatomical
microscopic characteristics features of below ground parts (root) for
correct identification and intrageneric and specific differentiation
among closely related species plants, which are very difficult to differ-
entiate through aerial parts laves and stem. Based on of root, these
information can be used as documented evidences for correct identifi-
cation record to create monograph of the plant. It is a simple and reli-
able tool, by which complete information of the crude drug can be
obtained and provide a meaningful way for the promotion of the tra-
ditional knowledge of the herbal medicines. In addition to this, it will
act as a tool to detect adulteration and substituents of pure drug and
will help in sustaining the purity, quality, reproducibility, and efficacy
of natural drug.
ORCID
Shafqat Ali Khan https://orcid.org/0000-0003-3740-3623
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How to cite this article: Khan SA, Barkatullah, Khan B.
Anatomy, micromorphology, and physiochemical analysis of
Rhus succedanea var. himalaica root. Microsc Res Tech. 2020;
1–12. https://doi.org/10.1002/jemt.23430