Research Paper Biochemistry Volume : 3 | Issue : 5 | May 2013 | ISSN - 2249-555X

Phytochemical Analysis, In-Vitro Antioxidant Activity

and Proximate Analysis on Rhinacanthus Nasutus(L)
Kurz Leaf

Keywords Phytochemical, R.nasutus, antioxidant, DPPH and H2O2.

Suman Bukke Venkataswamy Mallepogu ThyagaRaju Kedam

Department of Biochemistry Department of Biochemistry Department of Biochemistry
(Sponsored DST FIST & UGC BSR), (Sponsored DST FIST & UGC BSR), (Sponsored DST FIST & UGC BSR),
Sri Venkateswara University (DST- Sri Venkateswara University (DST- Sri Venkateswara University (DST-
PURSE) Tirupati– 517502, Andhra PURSE) Tirupati– 517502, Andhra PURSE) Tirupati– 517502, Andhra
Pradesh, India Pradesh, India Pradesh, India
ABSTRACT The present study provides an information on phytochemical, antioxidant and proximate analysis of different
extracts of Rhinacanthus nasutus leaf. Différent parts of R nasutus have been used in folk medicine for treat-
ing liver disorders, several skin diseases and other pharmacological effects. Different extracts of R.nasutus were prepared
based on the polarity in solvents of hexane, ethyl acetate, methanol and water for phytochemical analysis from leaves
of R.nasutus. The phytochemical analysis leaf extract has revealed the presence of flavonoids, triterpenoids, polyphenols,
steroids, saponins, alkaloids, carbohydrates, anthraquinones and least of tannins.
These principles at concentration of 250µg of preparation showed potential antioxidant properties as tested by the meth-
ods of radical scavenging activity, and methanolic extract showed more of peroxy radical scavenging property at all con-
centrations compared to hexane and ethyl acetate extracts. The Dried leaf powder Proximate analysis has revealed the
presence of 85% of dry matter, 13% of crude fiber, 11% of total ash, 1% of acid soluble ash and 4% crude protein. Hence
the present exploration of phytochemical antioxidants and proximate studies of R.nasutus, will be useful in the synthesis
and preparation of new drugs of pharmaceutical importance.

Introduction  Justiciinae subtype (Scotland and Vollesen, 2000). R nasutus

The identification of plants is useful to human beings from is widely distributed in some parts of sub-continent, in the
natural strands commenced in prehistoric studies. Experi- region of Southeast Asia and China (Farnsworth and Bun-
ments and trails with the two main ways through which hu- yapraphatsar 1992). The plant is a small slender shrub. The
mans have learnt various uses of the plants (Haseena Bhanu R nasutus is cultivated particularly as a medicinal plant has
et al., 2010 and Suman Bukke et al., 2011). In recent times, been used in treatments and preventions of diverse diseases
focus on plant research has increased all over the world and as folklore medicines. Different parts of the plant has used
a large body of evidence has collected to show immense po- in traditional medicine for the treatment in diseases such as
tential of medicinal plants used in various traditional systems. eczema, pulmonary tuberculosis, herpes, hepatitis, diabetes,
More than 13,000 plants have been studied during the last hypertension and several skin diseases (Siripong et al., 2006).
5 year period (Dahanukar et al., 2000). Over three-quarters The experimental evidences shows, that it has potential ef-
of the world population relies mainly on plants and plant fects for treatment of cancer, liver disorders, skin diseases,
extracts for health care. From plants the isolated and puri- peptic ulcers, helminthiasis, scurvy, inflammation and obesity
fied compounds in contrast, may lose their biological activity (Suja et al., 2003). The leaves of this plant are also used in the
or fail to behave in the same way as in the complex matrix preparation of shampoos. Rhinacanthine from roots induce
that the original item of food represents (Rao et al, 1998; apoptosis in human cervical carcinoma cells and hepatocellu-
Raveendra et al, 2008). According to the Food and Agricul- lar cancers (Wu et al.1998;Thirumurugan et al.,2000; Siripong
ture Organization (FAO), more than 50,000 plant species are et al., 2006 and 2009). Therefore considering the significant
used in the traditional folk medicine throughout the world importance of R.nasutus in folk medicine experiments were
(Schippmann et al., 2002). The drugs are derived from the conducted on analysis of plant products and their biological
whole plant or from different parts like leaves, stem, bark, activity.
root, flower, tuber and seed etc. More than 30% of the entire
plant species, at one time or other was used for medicinal Materials and Methods
purposes. It has been estimated that in developed countries Collection of plants and preparation of extracts:
such as United States, plant drugs constitute as much as 25% The R.nasutus were identified and authenticated by plant
of the total drugs, while in fast developing country such as Taxonomist, Department of Botany, Sri Venkateswara Uni-
India, the contribution is as much as 80% (Joy et al., 2001). In versity, Tirupati, Andhra Pradesh and voucher specimen no
19th century, medicinal plants and herbs were the soul source SVUBH/ 579. The fresh leaves of R nasutus were collected
of active principles capable of curing man’s aliments. Me- from Sesaschalam hills (Tirumala Hills and Tirupati) Chittoor
dicinal plants and herbs continue to be the source of proven district of Andhra Pradesh. Fresh leaves of R.nasutus (L) were
medicaments and of new and revolutionary drugs (Chatter- shade dried and milled to fine powder using a mechanical
jee 2000). In early 20th century new trend has emerged, as grinder. The powdered plant material was macerated with
several Botanists started surveying and identifying medicinal hexane, Ethyl acetate, methanol and water separately. The
plants on the basis to use in tribal and rural areas. Thus, the extract was then filtered with filter paper (Whatman No. 1)
economic importance of medicinal plants is much more to under reduced pressure using rota evaporator at 40°C. The
countries such as India than to rest of the world (Suman et concentrate was to obtain a dark molten mass then layered
al 2011) . on aluminum foil and freeze dried for further use.

The Rhinacanthus nasutus commonly known as “Rangchita Phytochemical screening

and Nagamalli” belongs to family Acanthaceae. The ge- Phytochemical examinations were carried out to detect the
nus Rhinacanthus comprises of about 25 species confined secondary metabolites (Alkaloids, saponins, carbohydrates,
to the Old World tropics and subtropics it is placed in the flavoinds, cardic glycosides etc.) in R.nasutus extracts by us-

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 Research Paper Volume : 3 | Issue : 5 | May 2013 | ISSN - 2249-555X

ing standard procedures/methods (Trease and Evans 1983; known to support bioactive activities in medicinal plants and
and Harbourne 1988). thus responsible for the antioxidant activities of this plant ex-
tract used in this study. The presence of flavonoids in the
DPPH (2, 2-diphenyl-1-picrayl hydrazyl) free radical scav- plants is likely to be responsible for the free radical scaveng-
enging assay ing effects observed. Flavonoids and plant phenolics are a
Evaluation of antioxidant activity was done by using 2, 2-di- major group of compounds that may act as primary antioxi-
phenyl-1-picrylhydrazyl (DPPH) method (Burits and Bucar dants or free radical scavengers (Polterait et al., 1997)
2000) with some modifications. Antioxidant reacts with DPPH
and convert it to α,α-diphenyl,-β-picryl hydrazine. One ml of Table-01: Phytochemical screening of different extracts of
plant extract was added to 4ml of 0.004% methanol solu- R.nasutus leaf
tion of DPPH. After 20-30 mins incubation period at room
temperature, DPPH solution with 2 ml of methanol was
used as sample (blank) and test samples we done at different S . S e c o n d a r y Hexane E t h y l Methanol Aqueous
No metabolites Extract Acetate
Extract Extract Extract
concentrations. The absorbance was read at against blank
at 517nm. Inhibition of free radical by DPPH in percent (1%)
was calculated by using the following equation. The degree 1 Steriods - + + -
of discolouration indicates the scavenging potentials of the
antioxidant extract. 2 Triterpenes - + + -

%DPPH radical/scavenging = [(∆ inAbsorbance of control- 3 Saponins - + + -

test sample) / (Absorbance of control)] x100
4 Alkaloids - + + -
Total antioxidant activity by Phosphomolybdenum Meth-
5 Carbohydrates - _ + +
od
The total antioxidant capacity of R.nasutus of different sol- 6 Flavonoids + + + +
vent extracts was evaluated according to the method, Prieto
et al.,1999. The absorbance of the samples were measured 7 C a r d i c- - - +
glycosides
at 695 nm in UV spectrophotometer. The higher absorbance
value indicates higher antioxidant activity. Ascorbic acid was 8 Anthroquinones - + + -
used as standard for comparison.
9 Polyphenols - + + +
Hydrogen Peroxide Method Note: “+” = presence and “-” = absence
The hydrogen peroxide assay was determined by the meth-
od Vijayabhaskaran et al 2010, with some modifications. DPPH (2, 2-diphenyl-1-picrayl hydrazyl) free radical scav-
A solution of hydrogen peroxide (20mm) was prepared in enging assay
phosphate buffer. To 2 ml of methanol and 2 ml of hydrogen The hexane, ethyl acetate, methanol and water extracts of
peroxide solution was added, and used as control. Methanol R.nasutus leaves were analysed for antioxidant property by
was used as blank and Ascorbic acid used as standard for using DPPH as recipient of radical. With increase in the range
comparison. After incubation for 10 minutes in dark, absorb- of 250-1000 μg/ml of extract, the antioxidant activity was
ance was recorded at 230 nm using UV-visible spectropho- found to be decreased in all extracts of hexane, ethyl ac-
tometer. etate, methanol and water when compared to BHT (Fig 01).
The ethyl acetate and methanol extracts showed the rela-
The H202 scavenging property was calculated using the for- tively highest actively compared to other preparations. The
mula: model of scavenging the stable DPPH radical is a widely used
method to evaluate the free radical scavenging ability of vari-
% Scavenging = (OD of Control – OD of Test / OD of Control) ous samples (Lee et al., 2003). Free radicals are involved in
× 100 many disorders like skin diseases, cancer and pharmacology
activity. DPPH is stable for free radical and is a sensitive way
Determination of Total phenolic assay to determine the antioxidant activity of plant extracts (Koleva
The total phenolic content of plant extract was determined et al., 2002 and Suresh et al., 2008)
by using the Folin-Ciocalteu assay (Singleton et al, 1965).
Gallic acid and double distilled water were used as standard Fig 01: Free radical (DPPH) scavenging activity of the
and blank. The reaction mixture was incubated for 90 mins at R.nasutus leaf extracts
room temperature, the absorbance against prepared reagent
blank was determined at 750nm with in UV-Vis Spectropho-
tometer. The total phenolic content was expressed as mg of
gallic acid equivalents (GAE)/100g fresh weight. All samples
were analysed in triplicates.

Proximate analysis
The Dried R.nasutus leaf powder was prepared for Proximate
analysis. It includes to prepare the Dry mater, total ash, Crude
protein, Crude fibre, Ether extract and Acid soluble in Ash.
The analysis was carried out using the AOAC methods 1990

Results and Discussion:

Phytochemical analysis
Preliminary phytochemical screening of the leaf extracts of
R.nasutus showed positive results for the presence of sec-
ondary metabolites like steroids, saponins, triterpinoids, al- Data are expressed as the mean of triplicate ± SD
kaloids, carbohydrates, flavanoids, polyphenols and glyco-
sides. Tannins are absent in all extracts prepared. Bioactive Total phenolic content
active compounds like steroids, alkaloids, carbohydrates, gly- The selection of solvents used for the extraction of phenolic
cosides, polyphenols were present in high amounts in metha- compounds depends on its solubility. It is well-known that
nol extract (Table-01) These phytochemical compounds are phenolic compounds contribute to quality and nutritional val-

INDIAN JOURNAL OF APPLIED RESEARCH X 33

 Research Paper Volume : 3 | Issue : 5 | May 2013 | ISSN - 2249-555X

ue in terms of modifying colour, taste, aroma, and flavor also Data are expressed as the mean of triplicate ±SD
in providing health beneficial effects. They also serve in plant
defense mechanisms to counteract reactive oxygen species Proximate analysis:
(ROS) in order to survive and prevent molecular damage and From our Proximate analysis of plant sample includes that
damage by microorganisms, insects, and herbivores (Vaya et the total ash is 11.4% in dried leaf , proteins shows the 4.46%
al., 1997). Phenols are very important plant constituents be- and fibre content in 13.24% in plant samples (Table 03) In this
cause of their scavenging ability due to their hydroxyl groups study the analysis provided an insight into the composition
and it involves directly to antioxdiative action (Hantano et of the tested medicinal plant in addition to their therapeutic
al., 1989). There were no correlation between total phenolic potentials. It was concluded that the presence of these nu-
content and antioxidant activity. Although some studies have trients and phytochemically-active components in the plant
demonstrated a correlation between phenolic content and sample might be responsible for their therapeutic activity
antioxidant capacity (Yang et al., 2002), our results (Table 02)
shows that methanol extracts contain highest antioxidant ac- Table 03: Proximate analysis of R.nasutus leaf powder
tivity among all the solvents. Hexane extract shows the least Dried R.nasutus
phenolic activity compared with all solvents. Total phenolic S. No. Proximate factors(Parameters) leaf powder (%
content was measured in conc. of total phenolics mg/ GAE/g w/w)
of extract. 1 Dry Matter 84.6 %
2 Total Ash 11.4 %
Table 02: Total Phenolic content and Phosphomolybde- 3 Crude Protein 4.46 %
num activity of the R.nasutus leaf extracts 4 Crude Fibre 13.24 %
5 Ether extract 0.74 %
Total Phenolic
content(Conc. of Phosphomolybdenum 6 Acid soluble in ash 1.06 %
Solvents total phenolics assay (mg AE/G of plant Conclusions
mg/GAEs/g of extract) The main focus of this work was to extract the plant active
extract
Hexane 19.6±1.9 57.06 ± 0.09 principles R.nasutus with different solvents, characterization
of the chemical constituents and evaluate the photochemi-
Ethyl Acetate 26.6±1.0 57.36 ± 0.16 cals and also antioxidant activities of extracts. Our stud-
Methanol 31.0±1.7 60.00 ± 0.02 ies provide a basis for various principles of R nasutus that
Aqueous 24.5±1.4 59.56 ± 0.00 are present and influence the free radical scavenging. Our
results on these extracts showed that the plant products
Phosphomolybdenum assay could serve as a good source for the therapeutic drugs for
The phosphomolybdenum method usually detects antioxi- degenerative diseases and exhibit good antioxidant po-
dants such as ascorbic acid, some phenolics, tocopherol, and tency as reflected by the results of the analysis performed
carotenoids (Prieto et al., 1999). Ascorbic acid, glutathione, and the components responsible for its efficacy identified for
cysteine, tocopherols, polyphenols, and aromatic amines its phytochemical nature. Among all the extracts prepared
have the ability to donate hydrogen and electrons. Generally, from R.nasutus leaves the bioactive compounds present are
aqueous or alcohol is considered the best solvent for extract- steroids, alkaloids, carbohydrates, glycosides, and polyphe-
ing phenolic compounds from plant materials (Negi et al., nols in high amounts. With regard to antioxidant activity
2003). Our results among hexane, ethyl acetate, methanol all methanolic extractions have showed and confirmed by
and water, the methanol shows the best antioxidant activity DPPH assay method. The hydrogen peroxide shows less in-
and the hexane shows the lowest activity (Table 02). Gallic hibitory effect but in case of assay hexane, ethyl acetate ex-
acid was used standard for this experiment. The plant ex- tract showed less antioxidant activity compared to methanol
tracts electron-donating capacity and thus they may act as extract. The results total phenolic and Phosphomolybdenum
radical chain terminators, transforming reactive free radical activity shows that methanol extracts shows highest among
species into more stable non-reactive products. The antioxi- all the solvents hexane shows the less activity compared with
dants break the free radical chain by donating a hydrogen all solvents. In this study the analysis provided an insight
atom (Gordon 1990 and Dorman et al, 2003). into the composition of the tested medicinal plant in addi-
tion to their therapeutic potentials. It was concluded that the
Hydrogen peroxide method presence of these nutrients and phytochemically-active com-
The hydroxyl radical is the most reactive free radical formed ponents in the plant sample might be responsible for their
in biological systems and is considered to be one of the therapeutic activity. The presence of the identified phyto-
quick initiators of the lipid peroxidation process Hydrogen chemicals makes the leaves pharmacologically active. Their
peroxide is weak oxidizing agent that inactivates a few en- antioxidant activity may be responsible for their usefulness
zymes directly, usually by oxidation of essential thiol (-SH) in the management and treatment of various diseases. The
group. From the Fig 02 The H2O2 was calucated in different proximate analysis the leaf nutrients in plants that are useful
concentrations in triplicates form that the increased in con- for many pharmacological activity. We are currently studying
centration decreases the values as the hexane values were other possible mechanisms of action of these leaves. Efforts
decreased than methanol and aqueous this shows the stabil- to identify the constituent compounds responsible for this
ity of activity. antioxidant activity are also in progress

Acknowledgments: The authors are thankful to UGC MRP

and DRDO to carry out this research and Prof. Kedam Thya-
gaRaju is a recipient of UGC BSR One time grant.

Fig 02: Hydroxyl radical scavenging activity of R. nasutus

leaf extracts

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