Abstract
Medicinal plants, such as Polyalthia longifolia (Indian mast tree), are important therapeutic sources for curing human diseases. In this work P. longifolia leaf extract was characterized by chromatographic and spectral fingerprinting techniques, phytochemical and heavy metal analyses, and microscopy. Light microscopy of a transverse section of the leaf of P. longifolia revealed the presence of various plant cells. Phytochemical screening results revealed the presence of alkaloids, triterpenoids, tannins, saponin, anthraquinones, and glycosides in the extract. The concentrations of heavy metals determined in the extract were well below the permissible limit. Nine peaks observed in the HPLC spectra showed the presence of various compounds in the extract. The GCMS method used for quantification of (3β,4α,5α,9β)-4,14-dimethyl-9,19-cycloergost-24(28)-en-3-yl acetate (i.e., cycloeucalenol acetate) in the extract was rapid, accurate, precise, linear (R2 = 0.8752), and robust. The HPTLC analysis showed ten specific peaks for the methanolic extract of P. longifolia leaf. Twelve major peaks in the range of 4,000 to 500 cm-1 were observed in the FTIR spectra, which represented various specific functional groups in the extract.
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Chromatographic and Spectral Fingerprinting of Polyalthia longifolia, a Source of Phytochemicals
Subramanion L. Jothy,a Chen Yeng,b and Sreenivasan Sasidharan a,*
Medicinal plants, such as Polyalthia longifolia (Indian mast tree), are important therapeutic sources for curing human diseases. In this work P. longifolia leaf extract was characterized by chromatographic and spectral fingerprinting techniques, phytochemical and heavy metal analyses, and microscopy. Light microscopy of a transverse section of the leaf of P. longifoliarevealed the presence of various plant cells. Phytochemical screening results revealed the presence of alkaloids, triterpenoids, tannins, saponin, anthraquinones, and glycosides in the extract. The concentrations of heavy metals determined in the extract were well below the permissible limit. Nine peaks observed in the HPLC spectra showed the presence of various compounds in the extract. The GCMS method used for quantification of (3β,4α,5α,9β)-4,14-dimethyl-9,19-cycloergost-24(28)-en-3-yl acetate (i.e., cycloeucalenol acetate) in the extract was rapid, accurate, precise, linear (R2 = 0.8752), and robust. The HPTLC analysis showed ten specific peaks for the methanolic extract of P. longifolia leaf. Twelve major peaks in the range of 4,000 to 500 cm-1 were observed in the FTIR spectra, which represented various specific functional groups in the extract.
Keywords: Fingerprinting techniques; P. longifolia; Heavy metal; Phytochemical; Characterization
Contact information: a: Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia; b: Dental Research & Training Unit, and Oral Cancer Research and Coordinating Centre (OCRCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia; *Corresponding author: srisasidharan@yahoo.com
INTRODUCTION
Plant-based therapy has been used as a vital component in traditional medicine systems and also serves as the main source of inspiration for several major pharma-ceutical drugs used in the defence against various diseases. The therapeutic potential, including the antioxidant, antimicrobial, and anticarcinogenic properties of higher plants, is due to the presence of secondary metabolites (Canigueral et al. 2008; Kaur and Arora 2009). Unsurprisingly, the human population has depended on medicinal plants for protection against diseases since time immemorial. Thus, the search for therapeutic agents and bioactive compounds to fight against emerging and existing diseases is an ongoing process, and medicinal plants have regularly been included as part of this search because they are rich in various bioactive compounds with useful medicinal properties. In consideration of the commercialization of formulations based on medicinal plants, the quality control standards of various medicinal plants that are used in traditional medicine are becoming more important. The characterization of extracts is of great importance to guarantee their quality. As a result, the current study was carried out for the evaluation of chemical components in Polyalthia longifolia.
P. longifolia belongs to the family Annonaceae. The tree is native to India and is locally known as Indian mast tree. P. longifolia is one of the most important indigenous plants in Indian medicinal literature; it is found throughout Malaysia and widely used in traditional medicine as a febrifuge and a tonic. Almost all parts of this plant are used in Indian medicine for the treatment of various ailments such as uterine disorders, fever, skin disease, diabetes, hypertension, helminthiasis, gonorrhea, and vitiated conditions of vata and pitta, and its significant medicinal properties have been reported in the scientific literature (Rastogi and Mehrotra 1960; Saleem et al. 2005; Katkar et al. 2010). Herbal Uterine Tonic manufactured by Tan Healthcare, India is an available marketed formulation which contains P. longifolia. The leaves possess anticancer potential, in which the bioactive compounds act through different pathways, converging, ultimately, into the activation of apoptosis in cancer cells, which leads to cell cytotoxicity (Verma et al. 2008). The stem bark, roots, and leaves have been studied for various biological activities, such as antibacterial, antidiabetic, anti-inflammatory, and antioxidant activity (Goutam et al. 2010; Nair et al. 2007; Ramakrishna et al. 2000; Chang et al. 2006). In addition, the bioactive compounds of this plant and its pharmacological activities due to its geographical diversity are relatively new areas for investigation.
Traditional medicinal plants such as P. longifolia have been widely used for thousands of years in various traditional medicine systems, such as those of the Ayurvedic, Chinese, Unani, Tibetan, Amazonian, and African cultures. However, one of the characteristics of medicinal plant preparations is that all the herbal medicines are extracted with boiling water (i.e., the decoction process) with one or many herbs. This may be the major explanation why the quality control of medicinal plants is extremely important. For this reason, the quantity and quality of the data concerning the safety and efficacy of medicinal plants play an important role in supporting their utilization in the treatment of various diseases. Accordingly, it seems to be essential to identify most of the phytochemical constituents of medicinal plants to ensure the reliability and repeatability of pharmacological and clinical research, to understand their bioactivities and the possible side effects of active compounds, and to enhance product quality control (Yan et al. 1999; Raven et al. 1999; Xie 2001). Therefore, several modern chromatographic techniques, such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectroscopy (GC–MS), infrared spectroscopy (IR), and high-performance thin layer chromatography (HPTLC), can be useful for this kind of quality control documentation. In this way, the full medicinal plant extract could be considered the active ‘compound’. The chromatographic fingerprint of the medicinal plant can be defined as the chromatographic pattern obtained using various modern chromatographic techniques on common phytochemical components of the extract. Normally, this chromatographic fingerprint, which is derived from the fundamental attributes of “integrity” and “fuzziness” or “sameness” and “differences,” enables the plant material under investigation to be characterized chemically (Xie 2001; Welsh et al. 1996). Therefore, chromatographic fingerprints of the medicinal plant extract can be useful for the authentication and identification of herbal preparations because no chemical characteristic of the phytochemical components of the extract is exactly the same for different sample preparations. Furthermore, the chromatographic fingerprints could demonstrate “variations” between various samples (Xie 2001; Valentão et al. 1999). In this study, we focused our investigation on how to construct a good chromatographic fingerprint for efficient evaluation of P. longifolia samples for the purpose of quality control. As a single herbal plant may consist of many phytochemicals, the fingerprints recorded by chromatographic instruments may present a relatively good integral representation of various phytochemicals of P. longifolia; this is the main topic of concern in this study. Thus, the characterization of P. longifolia extracts helps establish quality assurance for this plant. The present investigation was conducted for the characterization of P. longifolia using microscopic characteristics, phytochemical analysis, and heavy metal content analysis with chromatographic and spectral fingerprinting.
EXPERIMENTAL
Chemicals and Reagents
Chemicals and reagents were of analytical grade, purchased from either Sigma Chemical Co. (St. Louis, MO, USA) or Merck (Darmstadt, Germany).
Plant Sample Collection
The leaves of P. longifolia were collected from various areas in Universiti Sains Malaysia in Penang, Malaysia in January 2012 and authenticated at the Herbarium of the School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia, where a standard sample was deposited (Voucher specimen: USM/HERBARIUM/11306). The leaves were separated and cut into small pieces, which were first washed with tap water and then with distilled water. The leaves were then dried in an oven at 60 °C for 7 days, after which the dried leaves were ground into a fine powder using a grinder. The ground powder was stored in clean airtight bottles that were labelled.
Microscopic Studies and Powder Analysis
The microscopic features of the fresh leaves were determined using the method of Evans (1996). Histomorphological examination of P. longifolia leaf specimens was used to identify the histological microstructure. Leaf tissues were fixed in 10% buffered formalin. After fixation, the tissues were dehydrated in a graded series of alcohols, cleared with xylene, and embedded in paraffin wax. Multiple 5-mm microtome sections from the block were mounted on slides, stained with 0.5% methylene blue, and examined under a light microscope. For powder microscopy analysis, powder of P. longifolia leaf was mounted on a glass slide and observed under a light microscope (World Health Organization 1992).
Solvent Extraction
The dried sample (approximately 100 g) was added to methanol (300 mL) and soaked for 4 days at room temperature (30 ± 2 °C). The suspension was stirred to allow the leaf powder to fully dissolve in the methanol. Removal of the sample from the solvent was done by filtration through cheesecloth followed by filter paper (Whatman No. 1); the filtrate was concentrated under a vacuum to one-fifth its volume using a rotary evaporator at 60 °C and then sterilized by filtration using a 0.22-mm membrane. The thick paste obtained was further dried in an oven at 40 °C. The resultant extract was kept at 4 °C for further analysis. Methanol was used for extraction to mimic the usage of water by the traditional healers to prepare plant extracts as a decoction. Water and methanol have high dielectric constants and dipole moments as polar protic solvents. Moreover, the use of methanol makes the process of evaporation easier when compared to water.
Phytochemical Analysis
The leaf extracts of P. longifolia were screened for the following phytochemicals: saponin, tannins, steroids, flavonoids, alkaloids, triterpenoids, glycosides, and anthra-quinones, using the standard procedures recommended by Evans (1996) and Parekh and Chanda (2007).
High Performance Liquid Chromatography (HPLC) Analysis
The methanolic leaf extracts were further subjected to a reverse-phase HPLC analysis. The HPLC equipment consisted of a Shimadzu LC-6A pump, a Rheodyne injector fitted with a 20-µL loop, and a Shimadzu SPD-10AV UV-VIS with a variable wavelength detector from 190 to 400 nm. The chromatographic analyses were performed on a Novapak C18 column (4.6 x 250 mm) at ambient temperature with a mobile phase of methanol/water/formic acid (49:50:1, v/v) at a flow rate of 1 mL min-1. Detection was made at 190 to 400 nm. The data were acquired, stored, and analysed utilising Winchrom software.
Gas Chromatography-Mass Spectrometry (GC/MS) Analysis
The GC/MS analysis was done on a gas chromatograph-mass spectrometer (model Shimadzu 2010) equipped with a DB-5 capillary column (30 m long. 0.25 mm i.d., film thickness 0.25 µm). The column temperature programme was 60 ºC for 0 min, with 10 ºC per min increases to 270 ºC, for which the temperature programme was 30 min. The carrier gas was helium at a flow rate of 1 mL/min with a split ratio of 10:1. The detector and injector temperatures were both maintained at 270 ºC. The quadrupole mass spectrometer was scanned over the range 28 to 400 amu at 1 scan s-1, with an ionising voltage of 70 eV, an ionisation current of 150 mA, and an ion source temperature of 200 ºC. To determine the Kovats index of the components, a mixture of alkenes (C9-C24) was added to the crude extract before injecting it into the GC-MS equipment and analysed under the same conditions as above. The compounds were identified by a computer search from the commercial libraries of the National Institute of Standards and Technology (NIST) (Torey et al. 2010).
Fourier Transform Infrared (FTIR) Analysis
The methanol extracts of P. longifolia were mixed with KBr salt, using a mortar and pestle, and compressed into a thin pellet. Infrared spectra were recorded on a Shimadzu FTIR Spectrometer 8000 series, between 4,000 and 500 cm-1. All determinations were performed in triplicate (Torey et al. 2010).
HPTLC Finger Print profiles for P. longifolia Leaf Extract
TLC condition
A TLC plate of 20 × 10 cm was pre-coated with silica gel 60F254 TLC plates (E. Merck) (0.2-mm thickness) with an aluminum sheet support. The spotting device was a CAMAG Automatic TLC Sample Spotter (Camag Muttenz, Switzerland); the syringe volume was 100 μL; the developing chamber was a CAMAG Glass Twin Trough chamber (20 × 10 cm); and the densitometer consisted of a CAMAG TLC Scanner 3 linked to WINCATS software. The mobile phase was toluene: ethyl acetate: formic acid: methanol (3: 3: 0.8: 0.2, v/v). The saturation time for the mobile phase was 2 h.
Procedures
The plates were developed in a toluene: ethyl acetate: formic acid: methanol (3: 3: 0.8: 0.2, v/v) solvent system to a distance of 8 cm. The plates were dried at room temperature in air. The spots on the TLC plates were scanned at UV-Vis wavelength of 365 nm. The Rf values and the colour of the resolved bands were noted, where Rf (retention factor) values describe the distance traveled by the band divided by the distance traveled by the solvent.
Heavy Metal Analysis
Atomic absorption spectrometry (AAS) was used for the determination of the amount or concentration of specific heavy metals. AAS uses the phenomenon that atoms in the ground state absorb light of a specific wavelength that is characteristic of the particular atom when the light passes through an atomic vapour layer of the element to be determined (Ghaedi et al. 2008). The determination of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) were performed on a Perkin-Elmer 200 atomic absorption spectrophotometer under optimised measurement conditions using hollow cathode lamps according to the standard method in the British Pharmacopoeia (2011).
RESULTS AND DISCUSSION
Microscopy of Leaf
Transverse section
In general, the methods for quality control of medicinal plants involve sensory inspection (such as microscopic examinations) and analytical inspection using instrumental techniques (Choi et al. 2002). Accordingly, the current research commenced with the microscopic examination of a transverse section of the P. longifolia leaf. The advancement of the microscopy technique could help to achieve the goal of evaluation of the herbal products, particularly as the authentication methods of medicinal plants have been enhanced by the microscopic identification method. The Light Microscopic Method (LMM) for identification of medicinal plants has been applied for many years and has been published by Chinese Pharmacopoeia, as well as in other reference books, such as Chinese Materia Medica, New compendium of Chinese Materia Medica, British Herbal Pharmacopoeia, American Herbal Pharmacopoeia, Japanese Pharmacopoeia, Korea Herbal Pharmacopoeia, and Indian Ayurvedic Pharmacopoeia (Zhao et al. 2005). Moreover, the microscopic approach utilizes techniques, such as light microscopy, that facilitate analysis of the characteristics such as the presence or absence of particular cell types, that help to distinguish between the desired plant species and plant parts at the ultrastructural level. Thus, in this research, microscopic evaluation of the leaf material was performed, as the leaf extract was to be used in our subsequent study. A transverse section of the leaf of P. longifolia through the midrib was observed under a light microscope, which showed bowl-shaped abaxial parts and a straight adaxial side. Both the adaxial and abaxial epidermal layers were single-layered and thin-walled cubical cells. The epidermal cells were wide, polygonal, and thin-walled; the walls were straight or slightly wavy. The lower epidermis was stomatiferous. The lower epidermal cells were smaller compared to the upper epidermal cells. The epidermal cells were followed by four to six layers of angular collenchyma cells on both sides. In the midrib region, the vascular bundle was encircled by a schlerenchymatous ring. The bundle sheath, xylem, and phloem were clearly visible (Fig. 1). The light microscopic method offered several advantages over conventional authentication of medicinal plants, including effectiveness, simplicity, and low cost, as well as being widely adopted as an official method in many international herbal pharmacopoeias, as mentioned earlier.
Leaf powder
The powder was observed to be green to brownish in color. The diagnostic features of the powder include xylem elements, pitted vessels, stone cell, parenchyma cells, stomata, fiber of leaf, crystal, and calcium oxalate crystals (Fig. 2)
Phytochemicals Analysis
Phytochemical screening of the P. longifolia extracts (Table 1) revealed that the crude extracts contained alkaloids, triterpenoids, tannins, saponin, anthraquinones, and glycosides. The phytochemicals tested are known to exhibit medicinal activity and physiological activity. Alkaloids are an important drug source and have been reported to possess antimicrobial, antioxidant, and cytotoxic activity (Rahman et al. 2009). Medicinally, tannins are used in antidiarrhoeal, haemostatic, and antihaemorrhoidal preparations. Saponins are glycosides of triterpenes, steroids, or steroidal alkaloids found in plants. They are useful in lowering cholesterol (hypocholesterolemic property), as antioxidants, and as antidiabetic and anti-inflammatory agents.
Fig. 1. Transverse section of the leaf of P. longifolia. SC: Sclerenchyma cell; XY: xylem; PH: Phloem
Table 1. Phytochemical Analysis of P. longifolia
+ Present; – Absent
Terpenoids are a large and diverse class of naturally occurring organic chemicals found in all classes of living organisms, while steroids display analgesic properties (Rupasinghe et al. 2003; Sayyah et al. 2004; Malairajan et al. 2006; Tamil Selvan et al. 2012). Glycosides are drugs used in the treatment of congestive heart failure and cardiac arrhythmia and are found as secondary metabolites in medicinal plants (Edeoga et al. 2005). Some anthraquinones have been reported to possess antiosteoporotic activity (Li et al. 2009).
This study revealed that the extract of P. longifolia contained many important phytochemical constituents with various medicinal properties. The presence of biologic-ally important phytochemicals in the P. longifolia extracts may contribute to their reported medicinal value and indicates that it is a potential source for the development of useful drugs.
Heavy Metal Analysis
Another crucial factor relating to the safety of medicinal plants is the presence of contaminants such as heavy metals. The levels of mercury, lead, arsenic, and cadmium in the P. longifoliaextract were determined using AAS. None of these heavy metals was detected as being above the permissible limits in the extract, with most being well within the acceptable limits (Table 2). According to WHO (2007), the maximum amounts of lead and cadmium in dried plant materials are 10 ppm and 0.3 ppm, respectively, which are based on the acceptable daily intake (ADI) values.
Table 2. Heavy Metal Concentration in P. longifolia Leaf Extract
