Comparative botanical and phytochemical evaluation of Calotropis procera Linn . and Calotropis gigantea Linn . Root

Article history: Received on: 06/06/2015 Revised on: 02/07/2015 Accepted on: 17/07/2015 Available online: 27/07/2015 The present study aimed to develop pharmacognostical and phytochemical descriptors (HPTLC) of Calotropis procera and Calotropis gigantea. β-sitosterol which is one of the common terpene content and a potent antioxidant, purgative, antispasmodic and expectorant, has also been studied through a simple and high-precision method using high performance thin layer chromatography (HPTLC). This may be utilized by pharmaceutical industries for quality evaluation, ensuring successful commercial exploitation of this drug. From the present study it has been observed that both Calotropis procera and C. gigantea have similar microscopic characteristics, physico-chemical parameters showed a little variation as total ash components and extractive values are little less in C. gigantea. HPTLC studies also showed similar qualitative profile with some quantitative variations in total β-sitosterol, which was higher in C. gigantea (2.79%).


INTRODUCTION
Calotropis procera Linn.and Calotropis gigantea Linn.(Family: Asclepiadaceae) known, as 'Ark or Aak' in Ayurveda are versatile medicinal plants used singly or in combination with other medicinal plants for treating a variety of ailments like irritation, different types of pain, leprosy, syphilis, intestinal worms, cough, and it also act like emetic, diaphoretic, alterative, purgative and antispasmodic activity (Caius, 1986;Das, 1996).C. procera possesses potent antioxidant and anti-inflammatory properties and has been evaluated for its hepatoprotective effect against carbon tetrachloride (CCl 4 ) induced hepatotoxicity in rats (Padhy et al., 2007).The hypoglycaemic property of C. procera has also been assessed by an oral glucose tolerance test (OGTT) in streptozotocin-diabetic rat's (Bauer et al., 1966).The root of C. procera is used as a carminative in the treatment of dyspepsia (Kumar and Arya, 2006).Further, the root barks and leaves of Calotropis species have been used by various tribes of Central India as curative agents for jaundice (Samvatsar and Diwanji, 2000).The aqueous extract of the latex has been shown to inhibit cellular infiltration and provide protection against development of neoplastic changes in the transgenic mouse model of hepatocellular carcinoma (Choedon et al., 2006).The chloroform extract of the root exhibits protective activity against CCl 4 induced liver damage (Basu et al., 1992).
On chemical studies, flavonoids (Chopra et al., 1956;Singh andRastogi, 1972), triterpenoids (Pal andSinha, 1980), volatile long chain fatty acids (Sen et al., 1992), glycosides and proteases (Kitagawa et al., 1992) have been isolated from the various parts of the plant Calotropis species.Due to the various uses of Caloptropis procera and C. gigantea, the present study was aimed to develop botanical and phytochemical descriptors for roots of both species.β-sitosterols is widely available and easy to detect and quantify among these species through HPTLC.Thus as one of the active constituent β-sitosterols, a major terpenes, has also been quantified through a simple and high-precision method using high performance thin layer chromatography (HPTLC), which may be one of the identifying parameter used by industries for quality evaluation.

MATERIALS AND METHODS
The roots of C. procera and C. gigantea were collected at flowering stage from Lucknow, India.Materials were authenticated, and deposited in the Institute (please name the institute) herbarium (LWG No. 92906, 93248).Roots were preserved in 70% alcohol for histological studies.Microtome sections were cut and stained with safranin and fast green and photographed with Nikon F70X camera (Johansen, 1940).Physicochemical and phytochemical studies viz.total ash, acid insoluble ash, extractive values, total sugar, starch and tannin contents were carried on the shade dried powdered materials as per method described in AOAC (Anonymous, 1984;Peach and Tracy, 1955).

Preparation of root extracts:
Air dried (45-55 0 C) powdered root of C. procera and C. gigantea (1.0 g) in triplicate were extracted separately with 3×10 ml petroleum ether extract.Extracts were concentrated under vacuum, re-dissolved in methanol, filtered and finally made up of 100 ml with petroleum ether extract prior to HPTLC analysis.

Chromatographic conditions
Chromatography was performed on Merk HPTLC precoated silica gel 60GF 254 (20x20 cm) plates.Petroleum ether solutions of samples and standard compound β-sitosterol of known concentrations were applied to the layers as 6 mm-wide bands positioned 15mm from the bottom and 15 mm from side of the plate, using Camag Linomat 5 automated TLC applicator with nitrogen flow providing a delivery speed of 150nl/s from application syringe.These conditions were kept constant throughout analysis of the samples.

Detection and quantification of β-sitosterol
Following sample application, layers were developed in a Camag twin trough glass chamber that had been pre-saturated with the mobile phase of Toluene: ethyl acetate: Methanol (8.5: 1.5: 0.5) till the proper separation of bands up to 8cm height.After development, layers were dried with a dryer and derivatised with anisaldehyde-sulphuric acid reagent and β-sitosterol was simultaneously quantified using Camag TLC scanner model 3 equipped with Camag Wincats IV software.Following scan conditions were applied: slit width 6x0.45mm; wavelength, 320nm; absorption-reflection mode.In order to prepare calibration curves, stock solution of β-sitosterol (1mg/ml each) was prepared and various volumes of these solutions were analyzed through HPTLC exactly as mentioned above, calibration curves of peak area vs. concentration were also prepared.β-sitosterol (yield, 0.534% dry basis) had following values: R f 0.49 and r 2 0.9899.

Macroscopic and microscopic characters of the C. procera and C. gigantea root
Macroscopic and microscopic characters of roots both the species viz.C. procera and C. gigantea were similar and there were no marked differences which could be projected as key identifying parameters (Table1 and Fig. 1 and 2).
Physicochemical studies thus indicate more potentiality of C. procera as compared to C. gigantean, containing higher amount of extractive values.

HPTLC Studies
A densitometric HPTLC analysis was also performed for the development of characteristic fingerprint profile, which may be used as markers for quality evaluation and standardization of the drug.The percentage of total β-sitosterol was estimated through HPTLC methods and was higher in roots of C. gigantea (2.79%) than in roots of C. procera (1.07%) at R f value 0.49 and r 2 0.9899 .
It is observed that, both Calotropis procera and C. gigantea have similar microscopic characteristics, physicochemical studies showed a little variation; ash components and total extractive values are less in C. gigantea.However, HPTLC studies also showed similar qualitative profile with minor quantitative variations in β-sitosterol content, which was higher in C. gigantea.

CONCLUSION
Above studies concludes that both the species have almost similar profile and falls in the range as mentioned in ayurvedic pharmacopoeia of India.Thus either of these species may be used in any formulations.These data may also be useful as supportive information for the exploitation of C. gigantea as a substitute to C. procera (ark).Physico-chemical and HPTLC parameters of both the species may also be useful to pharmaceutical industries for the authentication and batch to batch consistency of the commercial samples.

Table 1
Comparative Macroscopic and microscopic characters of the roots of Calotropis procera and C. gigantea