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Pharmacognostic, Phytochemical and Pharmacological Studies of Cassia roxburghii | OMICS International
ISSN: 2155-9538
Journal of Bioengineering & Biomedical Science

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Pharmacognostic, Phytochemical and Pharmacological Studies of Cassia roxburghii

Pallavi Kamarapu*

Department of Pharmacognosy, Vaagdevi College of Pharmacy, Kakatiya University, Warangal, Telangana, 506009, India

*Corresponding Author:
Pallavi Kamarapu
Department of Pharmacognosy
Vaagdevi College of Pharmacy
Kakatiya University, Warangal
Telangana, 506009, India
E-mail: pallavisudheer2008

Received Date: April 10, 2015; Accepted Date: April 23, 2015; Published Date: April 30, 2015

Citation: Kamarapu P (2015) Pharmacognostic, Phytochemical and Pharmacological Studies of Cassia roxburghii. J Bioengineer & Biomedical Sci 5:152. doi:10.4172/2155-9538.1000152

Copyright: © 2015 Kamarapu P. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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In the present study, the leaf of Cassia roxburghii and its powder were subjected to pharmacognostic evaluation in terms of macroscopic and microscopic evaluation. The powdered drug was subjected to extraction with various solvents such as petroleum ether, chloroform, ethyl acetate, methanol and aqueous extract by successive maceration. The methanolic and aqueous leaf extracts exhibited reducing power compared with ascorbic acid at similar concentrations. Based on the biochemical estimation of serum marker enzymes and histological study, all the extracts showed hepato protective activity and the maximum activity was seen in methanol extract at 200 mg/kg. The order of activity Me200>aq200>Me400>Aq400.


Pharmacognostic evaluation; Cassia roxburghii; Enzymes


The liver is the major organ in the body, contributing about 1/50 of the total weight of the body. It lies in the upper part of the abdominal cavity. More than 500 vital functions have been identified with the liver. The liver is important because a person’s nutritional level is not only determined by what he or she eats, but by what the liver processes. It is difficult to detect symptoms of liver metabolic imbalances. Some of the common disorders of the liver include cirrhosis, viral hepatitis, alcoholic liver disease, hemochromatosis, liver cancer, jaundice and drug induced liver damage. Beyond the treatment of liver disorders, everyday care of the liver lays a cornerstone for total body health. Naturopaths and others, who look beneath the symptoms of an illness to its underlying cause, often discover that the liver had a role to play. People can suffer for a long time from a liver ailment without knowing of it. The incredible complexity of liver chemistry and its fundamental role in human physiology is so daunting to researchers that the thought that simple plant remedies might have something to offer is astonishing and incredible [1-5].

Cassia species have important role in phytochemical and pharmacological research due to their excellent medicinal values. Different classes of natural products, possessing potent physiological and pharmacological activities have been isolated from cassia species and they include anthracen derivatives, flavonoids and poly sacharides. Some of these compounds have been shown to possess Considerable antimicrobial activity. Cassia species are well known in folk medicine for their laxative and purgative uses. They are also used for treating skin diseases such as ring worm, scabies, eczema and wounds [6].

Materials and Methods

Collection of plant material

Cassia roxburghii is a uncommon in cultivation. The plant material collected from madikonda local areas of Warangal, India. Its parts were botanically authenticated by Taxonomist, Department of Botany, Kakatiya University, Warangal, India. A voucher specimen (CV-028) was maintained in the Department of Pharmacognosy and Phytochemistry, Vaagdevi College of pharmacy, India

Macroscopic, microscopic and physical evaluation of plant material

Macroscopic, Microscopic and physical evaluation of plant material was done by performing organoleptic, T.S, ash and extractive values.

Preparation of plant material

Cassia roxburghii leaves were washed under tap water and were efficiently dried under shade for about one week and protected from deterioration. The shade dried leaves were grinded made into powder with the help of blender.



The leaf material was weighed (250 g) and extracted by maceration using the solvents petroleum ether, chloroform, ethylacetate, methanol and aqueous at room temperature in a glass container for 3 days. The material was stirred from time to time to ensure proper extraction. After 3 days, the contents of the container were filtered through muslin cloth and the filtrate was concentrated under reduced pressure below 500C, until a soft mass obtained and then preserved in a desiccator. Finally phytochemical screening was done by performing the different identification tests [7-9].

Reducing power method

Different concentrations of the extracts from (100 μg/ml-1000 μg/ ml) in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml pH 6.8) and potassium ferricyanide (2.5 ml 1%). Then incubated at 500C for 20 min. To this, trichloroacetic acid was added and centrifuged at 3000 rpm for 10 min.

Then the upper layer was added with distilled water (2.5 ml), FeCl3 (0.5 ml 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates increased reducing power.

H2O2 scavenging assay method

H2O2 (43 mM) was prepared in phosphate buffer saline (pH 7.4). Positive control (Ascorbic acid) and extract solutions were prepared at concentrations of 50-250 μg/ml. Aliquots (different concentrations) of standard and extracts solutions (3.4 ml) were added to 0.6 ml of H2O2 solution. The reaction mixture was incubated at room temperature for 10 min and the absorbance was determined at 230 nm [10-13].

The % of scavenging was calculated as follows: % H2O2 scavenging = 100 X (absorbance of control – absorbance of sample)/absorbance of Control.

Acute toxicity testing

Acute toxicity and gross behavioral studies was carried out in mice after administration of various extracts of leaf of Cassia roxburghii. Albino mice weighing 20-25 gm were selected, weighed and marked. The mice were kept on overnight fasting before going to the test. The mice were divided into five groups with each group containing 6 mice. The test extracts were given orally in the form of suspension in arachis oil. The five groups of mice received the doses of extracts at 200, 400, 800 and 2000 mg/kg. Then the mice were continuously and carefully observed for 2 hrs followed by occasionally for further 4 hrs. The behavior and mortality of mice was observed up to 24 hrs. A one week washout period was allowed after studying each extract. Observation of behavioral changes will guide to go for further screening for the proposed activities [14].

Hepatoprotective Activity

In the present study, the animals were pretreated with test extracts and a standard drug silymarin (100 mg/kg) before inducing liver damage with CCl4. The duration of the study was seven days. After acclimatization the rats were divided into thirteen groups (I-XIII). Each group consisting of six animals. All animals were kept on same diet for 7 days. The division of animals for Hepatoprotective activity of leaf extract of Cassia roxburghii is as follows.

Group I served as normal and received 1 ml/kg of arachis oil p.o. for seven days. Group II served as toxic control and was given 5 ml/kg of 50% v/v CCl4 in olive oil i.p. on the seventh day. Group III (standard) animals were administered with 100 mg/kg of silymarin p.o. for seven days, followed by CCl4 administration i.p. on the seventh day. Group IV–VII were treated in a similar way to that of group III (standard) using methanol extract and aqueous extract of leaf of Cassia roxburghii at doses of 200 mg/kg and 400 mg/kg in place of standard respectively, followed by CCl4 administration i.p. on seventh day. All the rats were anaesthetized with thiopentone sodium (60 mg/kg i.p.) 36 h after administration of CCl4. Then blood was collected from common carotid artery by carefully opening the neck region of the rat. After blood collection, the blood samples were allowed to coagulate at room temperature for at least one hour. Serum was separated by centrifugation at 3000 rpm for 30 minutes and then analysed for total bilirubin, Lactate dehydrogenase (LDH), Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT), ALP (Alkaline phosphatase), total protein and albumin levels. The animals were then dissected and the livers were carefully removed and washed with 0.9% saline solution and preserved in formalin solution (10% formaldehyde) for histopathological studies [15-18].

Processing of Liver Tissue

Liver tissues were taken out from fixing solution and dehydrated for 30 minutes each in 30, 50, 70, 90, and 100% alcohol successively. To remove the alcohol from the dehydrated tissues, they were kept for 30 minutes each in alcohol: xylene (1:1) followed by pure xylene. Then the tissue were then kept in xylene : paraffin wax mixture (1:1) for 1 hour and then in molten paraffin wax at 62°C, after which they were trimmed and mounted on wooden blocks for thin sectioning. Hand microtome (York precision rotary microtome, model no YS1114) was used to cut thin sections of liver tissues of 5 μm thickness [19,20].

Staining and mounting of liver tissues

Ribbons of thin sections of liver tissues were placed in rows on clean glass slides previously coated with albumin-glycerine mixture and few drops of water added to let the sections float. The slides were heated on hot plate to fix liver sections onto the slides. The slides were then placed for 5minutes each in xylene to remove wax, then in absolute alcohol to remove xylene from the liver sections. Hydration of liver sections was attained by keeping them in descending series of alcohol and water mixtures (90%, 70%, 50%, 30% alcohol and in pure water) for three minutes each. Hydrated sections were stained with haemotoxylin stain for one minute and washed in running tap water to remove excess stain. Liver sections were dehydrated again by keeping in ascending of alcohol water mixtures (30%, 50%, 70%, and 90% alcohol) for one minute. After that, the sections were kept for 5 minutes each in absolute alcohol and then in xylene. Finally, the stained liver sections were mounted in DPX (Desterenedibutyls phthalate xylene) and viewed under optical microscope for histological examination [21-24].

Results and Discussion

In this evaluation macroscopy and microscopy of leaf of Cassia roxburghii were studied. The observations of the investigations were, the leaf powder of the plant was studied for their organoleptic characters like colour, odour and taste. The results of this study were Color – Greenish, Taste –mucilaginous, Odour - Characteristic

The leaf powder of the plant has shown the presence of following plant tissue systems under microscopic evaluation:

Calcium oxalate crystals : Prismatic type

Starch grains : Simple, compound

Vascular tissue : Xylem and phloem

Trichomes : Unicellular covering trichomes

Quantitative microscopy of leaf/leaf powder Cassia roxburghii: The powder analysis of leaf powder of the plant was evaluated and the results obtained were shown below.The width range of phloem fibers in powdered leaf of Cassia roxburghii was found to be 15.46 μ. The diameter of starch grains in powdered leaf of Cassia roxburghii was found to be 2.45 μ (Simple and compound). The length of the calcium oxalate crystals in powdered leaf of Cassia roxburghii was found to be 3.2 μ. Vascular bundle: Xylem and Phloem (Figures 1-3).


Figure 1: Powder analysis.


Figure 2: Trichomes.


Figure 3: Epidermis.

Transverse section of leaf

Rubiaceous or Paracytic stomata.

Spongy tissue of parenchymatous cells.

Cluster crystals of calcium oxalate (prismatic type) in palisade and crystal sheath in mid rib region.

Palisade-a single layer below upper and lower epidermis.

Epidermal trichomes-Unicellular covering trichomes.

Epidermis-beaded walled epidermis (Figure 4 and Tables 1-3).

Particulars Leaf powder (%w/w)
Total ash 16
Acid insoluble ash 0.59
Water soluble ash 1

Table 1: Physicochemical evaluation.

S.No Part of plant Method of extraction Solvent Physical
% Yield
1 Leaf Maceration Pet. ether Resinous 1.42
2 Leaf Maceration Chloroform Resinous 2.2
3 Leaf Maceration Ethyl acetate Resinous 2.1
4 Leaf Maceration Methanol Powder 2.1
5 Leaf Maceration Water Powder 3.2

Table 2: Percentage yield of the Cassia roxburghii leaf extracts

Tests Leaf  extracts
- Dragendorff’s test
- Mayer’s test
- Wagner’s test
- Hager’s test
- Molish’s test
- Fehling’s test
- Benedict’s test
- Barfoed’s test
- Liebermann-
Burchard reaction
- Salkowski test
Phenolics and Tannins
- 5% ferric chloride test
- Lead acetate solution test
-Borntragers test
-Keller killani test
-Saponin test
-Millons test
Xanthoprotein test

Table 3: Qualitative phytochemical screening of different extracts of Cassia roxburghii.


Figure 4: T.S of leaf.

Acute toxicity studies were performed for all the five extracts at doses of 200, 400, 800 and 2000 mg/kg body weight in mice. The behavioral changes were observed for 24 hours. The observation of the behavioral changes the animals did not show any toxic effects up to the dose of 2000 mg/kg.

Antioxidant activity

Reducing power method (Tables 4-6 and Figure 5): The reducing power of methanolic and aqueous extracts from cassia roxburghii compared with ascorbic acid as standard. The reducing power of both samples increased with the concentrations. The reducing power of methanolic, aqueous extracts were 1.01,0.55 at 1 mg/ml respectively however at 1 mg/ml ascorbic acid showed excellent reducing power of 0.91, respectively which are significantly higher than that of methanol and aqueous extracts. In the present study methanolic extract showed higher reducing power than Aqueous extract.

Ascorbic acid Concentration
Absorbance at 700 nm
100 0.26
200 0.42
400 0.60
600 0.72
800 0.84
1000 0.91

Table 4: Table showing ascorbic acid absorbance.

leaf aqueous extract
Absorbance at 700 nm
100 0.29
200 0.36
400 0.64
600 0.74
800 0.93
1000 1.01

Table 5: Table showing leaf methanol extract absorbance.

leaf aqueous extract
Absorbance at 700 nm
100 0.14
200 0.15
400 0.18
600 0.45
800 0.46
1000 0.55

Table 6: Table showing leaf aqueous extract absorbance.


Figure 5: Reducing power of the plant extract Cassia roxburghii.

H2O2 scavenging assay method (Tables 7-10 and Figure 6)

The methanolic and aqueous whole plant extracts exhibited higher H2O2 scavenging activity than ascorbic acid at similar concentrations. The IC50 values of the methanolic and aqueous extracts of leaves and ascorbic acid were 110.375, 136.599 and 136.633 μg/ml, respectively (Table 11 and Figures 7-10).

Ascorbic acid
Absorbance at 230 nm % Inhibition
50 0.146 16.04
100 0.26 28.5
150 0.422 46.3
200 0.818 89.8
250 0.91 100

Table 7: Table showing ascorbic acid % inhibition.

leaf aqueous extract concentrations (µg/ml) Absorbance at 230 nm % Inhibition
50 0.209 22.4
100 0.399 42.8
150 0.483 51.8
200 0.517 55.5
250 0.931 100

Table 8: Table showing leaf aqueous extract % inhibition.

Leaf methanolic extract concentrations (µg/ml) Absorbance at 230 nm % Inhibition
50 0.728 71.02
100 0.736 71.80
150 0.804 78.43
200 0.904 88.19
250 1.025 100

Table 9: Table showing leaf methanolic extract % inhibition.

S.No Particulars IC50 Values(μg/ml)
1 Ascorbic acid 136.633
2 Methanol extract of
Cassia roxburghii
3 Aqueous extract of
Cassia roxburghii

Table 10: Table showing IC50 values.

  SGPT SGOT ALP Bilirubin Protein Cholesterol
Control 45.08 ± 1.22 38.52 ± 0.51 174.03 ± 1.672 0.48 ± 0.019 4.53 ± 0.001 67.5 ± 0.191
Toxic (ccl4) 307 ± 8.38 389.3 ± 13.4 368 ± 2.8 0.83 ± 0.018 2.13 ± 0.024 34.45 ± 0.152
Standard (Silymarin) 43.64 ± 0.31 42.12 ± 0.20 182 ± 1.585 0.46 ± 0.006 5.26 ± 0.020 65.26 ± s0.138
Methanol 200 mg 54.26 ± 0.138 68.23 ± 0.157 136 ± 1.563 0.52 ± 0.012 6.81 ± 0.199 73.33 ± 1.202
Methanol 400 mg 64 ± 1.317 138.6 ± 0.112 266.6 ± 1.585 0.63 ± 0.010 4.7 ± 0.173 54.26 ± 0.138
Aqueous 200 mg 56.46 ± 0.140 75.06 ± 0.120 147 ± 1.555 0.53 ± 0.012 6.45 ± 0.125 64 ± 1.317
Aqueous 400 mg 73.33 ± 1.202 186.5 ± 0.002 336.48 ± 0.149 0.58 ± 0.014 5.56 ± 0.170 56.46 ± 0.140

Table 11: Effect of different leaf extracts of Cassia roxburghii on serum biochemical parameters in CCl4 induced liver toxicity.


Figure 6: H2O2 scavenging activity of plant extracts of cassia roxburghii.


Figure 7: Effect of different leaf extracts of Cassia roxburghii on ALP (U/L) in serum.


Figure 8: Effect of different leaf extracts of Cassia roxburghii on bilirubin in serum.


Figure 9: Effect of different leaf extracts of Cassia roxburghii on cholesterol in serum.


Figure 10: Effect of different leaf extracts of Cassia roxburghii on total protein in serum.

Histological study

Shown in Table 12 and Figures 11-14.

S.NO. Group Score Observation
1. Control 0 Showed normal hepatic cells with well preserved cytoplasm, nucleus, nucleolus and central vein.
2. CCl4 4 Showed necrosis, steatosis, fatty vaculaization and pseudolobule formation were seen.
3. Reference standard 0 Showed significant restoration of liver architecture comparably with normal rats
4. Methanolic
(200 mg/kg)
1 Showed almost normal liver architecture with very mild degree of fatty changes when compared with normal and standard.
5. Methanolic
(400 mg/kg)
3 Moderated restoration of liver architecture with mild degree of necrosis.
6. Aqueous
(200 mg/kg)
2 Showed mild fatty change and mild sinusoidal Congestion.
7. Aqueous
(400 mg/kg)
3 Showed mild degree of necrosis with areas of inflammation.

Table 12: Grading of liver damage based on histological study.


Figure 11: Toxic.


Figure 12: Control.


Figure 13: Reference standard.


Figure 14: Methanol (200 mg/kg).


Whole plant and seeds are used to treat skin diseases like ringworm, antimicrobial, hepatoprotectivity. The methanolic and aqueous leaf extracts both exhibited Antioxidant activity compared with ascorbic acid. Methanolic extract found to be more potent Antioxidant activity compare with aqueous leaf extract. All the leaf extract of Cassia roxburghii showed hepatoprotective activity and methanolic extract (200 mg/kg) was found to be more potent hepatoprotective compare with the other extracts. The histological studies also indicated that methanol extract (200 mg/kg) showed significant activity when compared with other extracts. Therefore, it can be concluded that Cassia roxburghii may have “potential hepatoprotective activity thus further mechanism based studies are needed for its hepatoprotective activity”.


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