Pharmacognostic and Phytochemical Evaluation of Chrysophyllum Cainito Linn
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Int. J. Pharm. Sci. Rev. Res., 26(1), May – Jun 2014; Article No. 17, Pages: 106-111 ISSN 0976 – 044X Research Article Pharmacognostic and Phytochemical Evaluation of Chrysophyllum cainito Linn. Leaves Sunita Shailajan*, Deepti Gurjar Herbal Research Lab, Ramnarain Ruia College, Matunga (E), Mumbai, India. *Corresponding author’s E-mail: [email protected] Accepted on: 16-02-2014; Finalized on: 30-04-2014. ABSTRACT Chrysophyllum cainito L. (Sapotaceae); commonly known as Star Apple is a tropical tree native to lowland of Central America and West Indies and exotic to the warmer parts of India. Leaves of C. cainito are reported to possess antidiabetic and antioxidant properties as well as have been used against articular rheumatism. A decoction of leaves is taken as pectoral. In the present study, macroscopic, physicochemical, phytochemical and safety profile of C. cainito leaves has been evaluated and discussed. Findings of this study can be useful as reference information in order to evaluate quality of C. cainito leaves. Keywords: Chrysophyllum cainito Linn., Gallic acid, Physicochemical evaluation, Safety evaluation, Triterpenoids. INTRODUCTION inflammatory, anticancer, antimicrobial, hrysophyllum Linn. (Sapotaceae), is a genus of hepatoprotective, cadioprotective activity, etc.8) and evergreen trees, distributed mainly in the tropics gallic acid (antioxidant, anti-allergic, anti-inflammatory, Cespecially America with a few species in West anti-mutagenic, anti carcinogenic, apoptotic and wound Africa and Australia. C. cainito is one of the species of this healing, etc.9); chromatographic characterization of C. genus which occur in warmer parts of India1 and is cainito leaves was carried out in terms of these commonly known as Star Apple. The plant is highly triterpenoids (ursolic acid, β-sitosterol and lupeol) and desired throughout the tropics as an ornamental tree and phenolic compound (gallic acid) content. Further, in order for the production of its large edible fruits.2 The ripe fruit, to evaluate safety of C. cainito leaves, acute oral toxicity because of its mucilaginous character, is eaten to sooth study (as per OECD guideline no. 420) was conducted in inflammation in laryngitis, pneumonia and given as a Albino Wistar rats. treatment for diabetes mellitus. The bark is considered as a tonic, stimulant and its decoction is used as an MATERIALS AND METHODS antitussive.1 Infusion of the leaves has been used against Chemicals and reagents diabetes, articular rheumatism and its decoction is taken as pectoral. C. cainito leaves have been chemically Ursolic acid, β-sitosterol, lupeol and gallic acid (98 % screened and reported to possess alkaloids, flavonoids, purity each) were procured from Sigma Aldrich Chemical phenols, sterols and triterpenes.3 Company, (Steinheim, Germany). Chemicals of analytical grade were purchased from Merck Specialties Private Pharmacognosy, in recent years has gained immense Limited, Mumbai. importance as it is an efficient tool for the authentication and identification of plant raw materials and therefore Plant sample evaluation of pharmacognostic parameters is an Fresh leaves of C. cainito were collected from two indispensible step when dealing with herbal drugs.4 A different locations of Mumbai (Sion and Ruia College thorough literature survey revealed that, though C. campus, Matunga). The taxonomic identification of a cainito plant is of high therapeutic value, scientific data representative sample was confirmed by Agharkar on pharmacognostic parameters of C. cainito leaves Research Institute, Pune (Authentication no. Auth 13- remains an unexplored issue. Hence, establishment of a 020). The plant sample was shade dried for a week pharmacognostic profile of C. cainito leaves will assist in followed by drying in an oven preset at 450C for 4 days. its standardization in terms of quality, purity and sample The samples were powdered in a mixer grinder, sieved identification.5 Therefore, the aim of this study was to through 85 mesh (BSS) and stored in an air tight standardize C. cainito leaves by carrying out macroscopic, containers. physicochemical, phytochemical, chromatographic and safety evaluation. Macroscopic evaluation Based on the reported therapeutic activities of ursolic Macroscopic characters of fresh leaves such as type of the acid (anticancer, anti-inflammatory, anti-oxidative and leaf base, presence or absence of petiole, characters of hepatoprotective, etc.6), β-sitosterol (anticancer, lamina, venation, margin, apex, base, surface and texture estrogenic, hypolipidemic, etc.7), lupeol (anti- were studied. International Journal of Pharmaceutical Sciences Review and Research Available online at www.globalresearchonline.net 106 © Copyright protected. Unauthorised republication, reproduction, distribution, dissemination and copying of this document in whole or in part is strictly prohibited. Int. J. Pharm. Sci. Rev. Res., 26(1), May – Jun 2014; Article No. 17, Pages: 106-111 ISSN 0976 – 044X Physicochemical evaluation analytical balance (Goettingen, Germany) was used to weigh the standard. Chromatographic separation of the The physicochemical parameters of the leaves such as phytochemical constituents was achieved on TLC plates foreign organic matter, ash content (total, acid insoluble (E. Merck) pre-coated with silica gel 60 F (0.2 mm and water soluble) and extractive values were 254 thickness) on aluminium sheet support. determined using standard Pharmacopoeial methods.10-11 To develop an HPTLC fingerprint of C. cainito leaves, the Phytochemical evaluation sample (10.0 µL) was applied to the plate as a band of 8.0 Phytochemical screening of some major secondary mm wide and at a distance of 15.0 mm from the edges. metabolites (flavonoids, essential oils, tannins, glycosides, Plate was developed up to a distance of 85.0 mm in alkaloids and resins) in C. cainito leaves was carried out by CAMAG twin trough glass chamber pre-saturated with the performing preliminary phytochemical tests as per the mobile phase chloroform: toluene (8: 2, v/v) for 15 min. methods reported12 and further the powder of dried C. After development, the plate was dried in a current of air cainito leaves was subjected to phytochemical evaluation at room temperature. The plate was derivatised using by successively soxhlet extraction with various organic 10% methanolic sulphuric acid and dried in oven preset at solvents in order to analyze the percent extract of major 110 0C for 10 min. For densitometric scanning, the source class of compounds present in the plant raw material as of radiation was a mercury lamp (366 nm). All per the method reported.13 measurements were performed at 22 ± 1oC. Plate was photo-documented at 366 nm. Chromatographic characterization For simultaneous separation of ursolic acid, β-sitosterol Extraction of phytochemical constituents from C. cainito and lupeol from C. cainito leaves, the sample was diluted leaves using methanol (1:1, v/v) and 10.0 µL of it along with the The powdered sample (0.2 g) was extracted with standards- ursolic acid (10.0 µg/mL), β-sitosterol (10.0 methanol (5.0 mL), vortex mixed for 1 min and sonicated µg/mL) and lupeol (20.0 µg/mL) of 10.0 µL each were for 20 min followed by filtration through Whatman filter spotted on TLC plate as bands of 8.0 mm wide and at a paper No. 1. The filtrate was subjected to HPTLC analysis distance of 15.0 mm from the edges under similar for the development of a phytochemical fingerprint. instrumental conditions. Plate was developed up to a distance of 85.0 mm in CAMAG twin trough glass In order to extract triterpenoids namely ursolic acid chamber pre-saturated with mobile phase toluene: (Figure 1A), β-sitosterol (Figure 1B) and lupeol (Figure 1C) methanol (8:1, v/v) for 15 min and derivatised using 10% from the complex matrix of C. cainito leaves, the methanolic sulphuric acid. Post derivatization procedure powdered sample (0.5 g) was extracted with petroleum 14 was kept similar for the development of fingerprint and ether (10.0 mL ), vortex mixed for 1 min and then estimation of these markers. sonicated for 20 min followed by filtration through Whatman filter paper No. 1. Filtrate was evapourated To separate gallic acid from C. cainito leaves, sample (10.0 under vacuum using rotary evaporator to dryness at 400 µL) and gallic acid standard (100.0 µg/mL, 10.0 µL) were C, reconstituted in equal volume of methanol and spotted on TLC plate as bands of 8.0 mm wide and at a subjected to HPTLC analysis for optimized separation of distance of 15.0 mm from the edges under similar these triterpenoids. instrumental conditions. Plate was developed up to a distance of 85.0 mm in CAMAG twin trough glass In order to extract a phenolic compound gallic acid chamber pre-saturated with mobile phase toluene: ethyl (Figure 1D), the powdered sample (1.0 g) was extracted acetate: formic acid (2:7:1, v/v/v) for 15 minute. The plate with methanol (10.0 mL), vortex mixed for 1 min and was scanned and photo documented at 254 nm. sonicated for 20 min followed by filtration through Whatman filter paper No. 1. The filtrate was subjected to Safety evaluation (acute oral toxicity study) HPTLC analysis for optimized separation of gallic acid. Acute oral toxicity15 of C. cainito leaves (aqueous slurry Preparation of standard stock solutions and ethanolic extract) was conducted and the results were compared with the animals of control group treated Standard stock solution of ursolic acid, β-sitosterol, lupeol with distilled water [guidelines: OECD guideline no.