Phaleria macrocarpa - Lincoln University College

Review Received: 27 May 2014

Revised: 4 November 2014

Accepted article published: 27 November 2014

Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4603

Bioactive compounds and advanced processing technology: Phaleria macrocarpa (sheff.) Boerl, a review Mst. Sabina Easmin,a Md. Zaidul Islam Sarker,a* Sahena Ferdosh,b Siti Hadijah Shamsudin,a Kamaruzzaman Bin Yunus,b Md. Salim Uddin,a Md. Moklesur Rahman Sarker,c Md. Jahurul Haque Akanda,d Md. Sohrab Hossaine and HPS Abdul Khalile Abstract Recent technological advances and the development of new methods has provided an opportunity to obtain highly purified natural bioactive compound extracts with potential for the treatment and prevention of human diseases. The use of hazardous and toxic solvents for the extraction and processing of bioactive compounds from plant materials is considered a problem for health, safety and environmental pollution. Advanced technology aims to increase production of the desired compounds and find an alternative to using toxic solvents in the extraction of bioactive compounds from plant materials. The ever growing interest in plant bioactive compounds and today’s concerns about environment issues have led to an increased need for an efficient and green extraction method. This review is focused on the extraction of bioactive compounds from plants using advanced and environment-friendly methods such as supercritical fluid extraction, microwave-assisted extraction, ultrasound-assisted extraction and similar techniques that can extract rapidly and free from organic residues. An updated overview of the bioactive compounds present in the plant Phaleria macrocarpa and its extraction, fractionation, purification and isolation is provided. The advantages and disadvantages of both conventional and non-conventional extraction methods are also discussed in this review. © 2014 Society of Chemical Industry Keywords: medicinal plant; Phaleria macrocarpa; extraction of bioactive compounds; conventional extraction method; non-conventional extraction method

INTRODUCTION At one time, plants were used as a source of food only. Nowadays, plants are widely used as a natural source of medicinal agents, food additives, cosmetics and neutraceuticals.1 In recent years, research on medicinal plants has received considerable attention. A large variety of structures and functionalities of natural bioactive compounds gives an excellent pool of molecules that produce essential functional foods, neutraceuticals, food additives and pharmaceuticals for human health benefits. Extracts from medicinal plants provide huge opportunities for new drug discoveries because of the structural complexity and unprofitability of chemical synthesis. Also, synthetic drugs have been associated with many side effects on human health.2 Environment-friendly and organic-residues-free extraction methods, termed green technology, for the extraction of bioactive compounds from medicinal plants are currently a most interesting research area. The majority of the extraction solvents are organic chemicals that are hazardous and toxic to health and environment. This is exacerbated by the high cost and ensuing environmental problems from the large amount of waste by-products from these chemical industries. Organic solvents produce greenhouse gases in the environment that are dangerous for humans, agriculture, microorganisms, etc. Of late, a mounting interest has developed for a safer and greener J Chem Technol Biotechnol (2014)

extraction approach including ‘green solvents’, ‘green processing’ and ‘green product’ to avert any health, safety and environmental consequences. Therefore, green chemistry is required to promote the idea of ‘greener’ solvents (non-toxic, benign to environment), for replacement in cases that can be substituted with safer alternatives, or changes in the methodologies of extractions such that

∗

Correspondence to: Md. Zaidul Sarker, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan Campus, 25200 Kuantan, Pahang, Malaysia. E-mail: [email protected] or [email protected]

a Faculty of Pharmacy, International Islamic University Malaysia, Kuantan Campus, 25200 Kuantan, Pahang, Malaysia b Faculty of Science, International Islamic University Malaysia, Kuantan Campus, 25200, Kuantan, Pahang, Malaysia c Clinical Investigation Centre, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia d Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, 56000 Kuala Lumpur, Malaysia e School of Industrial Technology, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia

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www.soci.org solvents are not needed. Limitations of conventional methods as well as the necessity for pure products in food, cosmetics, neutraceuticals and pharmaceuticals encourage efficient, green and alternative extraction methods. Green extraction methods could be defined as extraction processes that reduce energy consumption, allow use of alternative solvents or without solvents, and use of renewable natural sources ensuring a safe and high quality product. The non-conventional methods are considered as ‘green processing’. These methods are able to either cut down or reduce the consumption of organic solvents and degradation of samples, elimination of valuable materials of the sample, clean-up and concentration steps before chromatographic analysis, improvement in extraction efficiency, selectivity and/or kinetics of the extraction. The ease of automation of these techniques also favors their use for the extraction of plant materials.3 More than 80% of the world’s population depends on traditional medicine in treating ailments, as reported by the World Health Organization (WHO).3 Plants contain a wide range of compounds promising as traditional medicine to treat chronic as well as infectious diseases. Thousands of phytochemicals from plants have been considered safe and effective alternatives with fewer side effects. These phytochemicals, which are secondary metabolites of plants, include tannins, terpenoids, alkaloids, flavonoids and others, which have many biological remedial benefits such as anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic and wound healing. These bioactive compounds are used to develop either drugs or dietary supplements.1 Phaleria macrocarpa (Scheff.) Boerl is a popular medicinal plant in many south Asian countries. Traditionally, P. macrocarpa is used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and heart diseases, kidney disorders, blood related diseases, acne, stroke, migraine, and various skin ailments.1,4 Every part of this plant from leaves to roots is used for the prevention of diseases with some good results. The stems, leaves and fruits of P. macrocarpa have been widely used for medicinal treatments. The constituents of the stem are used to treat bone cancer; seeds are used in treating breast cancer, cervicle cancer, lung, liver and heart diseases; while the leaves are used to treat impotence, diabetes mellitus, allergies, tumors and blood diseases.5 This herb has been used in both unprocessed and processed form as tea, juice and other liquid forms. The unprocessed fruit may have toxicity and sometime are poisonous.6 Due to the side effects of synthetic drugs and the development of microbial resistance to chemically synthesized drugs, researchers are interested in the extraction, isolation and characterization of bioactive compounds from natural sources. The growing awareness of the health benefits has led to an interest in consuming foods enriched with bioactive compounds. The important steps needed to utilize bioactive compounds from natural sources are extraction, pharmacological screening, isolation and characterization, and toxicological and clinical evaluation.3 However, the toxicity of the solvents, the degradation of the compounds, consumption of time, total yield and selectivity of the process are major setbacks in the extraction process. These points are directly linked to environment pollution, economic viability, quality and purity of the final product.7 The limitations of conventional methods as well as the demand for organic residue-free products (green products) in food, cosmetics, neutraceuticals and pharmaceuticals highlight the need for efficient, green and alternative (non-conventional) extraction methods. In this review, chemical constituents, biological activities and the extraction of bioactive compounds from P. macrocarpa have been

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extensively discussed. From published reports it can be seen that extraction from this plant was carried out using only conventional extraction methods. The extraction of bioactive compounds from P. macrocarpa using non-conventional methods could provide its application in food, cosmetic, and pharmaceutical industries to obtain products free from organic residues. Phaleria macrocarpa and its chemical constituents Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae family) is commonly known as Mahkota Dewa, pau or God’s crown. This plant is called simalakama in Sumatra (Malay) and Depok (West Java, Indonesia). In Java, it is known as makuto rajo, makutadewa, makuto ratu or makuto mewo.8 Phaleria macrocarpa grows throughout the year in tropical areas, reaching a height of between 1 and 6 m. It is a complete tree consisting of stem, leaves, flower and fruit, the fruit being elliptical with a diameter of around 3 cm. The color of the fruit is green before ripening and turns red when fully ripe. A photograph of the plant and fruit of P. macrocarpa is shown in Fig. 1. The different parts of P. macrocarpa contain mahkoside-A, dodecanoic acid, palmitic acid, des-acetyl flavicordin-A, flavicordin-A, flavicordin-D, flavicordin-A glucoside, ethyl stearate, lignans and sucrose.9 Mahkoside-A (4,4′ dihydroxy2-methoxybenzophenone-6-O-𝛽-D-glucopyranoside) was first isolated from the pit of P. macrocarpa along with six other known compounds including magniferin, kaempferol-3-O-𝛽-D-glucoside, dodecanoic acid, palmitic acid, ethyl stearate, and sucrose.10 Lignans such as pinoresinol (79 ± 4% [−] -enantiomer excess), lariciresinol (55 ± 6% [−] -enantiomer excess) and matairesinol (pure [+] -enantiomers) were also isolated from different parts of P. macrocarpa by chiral column analysis.11 Saponins, alkaloids, polyphenolics, phenols, flavanoids, lignans and tannins are found in the bark and fruit.12,13 Fruit contains icariside C3 , magniferin, and gallic acid.2,11,14 Phalerin was first isolated from leaves of P. macrocarpa as benzophenone glycoside (3,4,5-trihydroxy-4-methoxy-benzophenone-3-O-𝛽-D-glucoside).15 Phalerin was also isolated from fruit, however, the proposed structure (2,4,6-trihydroxy-4-methoxy-benzophenone-3-O-𝛽-Dglucoside) was slightly different from the previous report (3,4,5-trihydroxy-4-methoxy-benzophenone-3-O-𝛽-D-glucoside).14 Kaempferol, myricetin, naringin and rutin are obtained in the pericarp of the fruit and the mesocarp and seed contain naringin and quercitin.2 Phorboesters, des-acetyl flavicordin-A and 29-norcucurbitacin derivatives have been isolated from seed. The main chemical constituents of the oil extracted from seed were oleic acid and linoleic acid as the main fatty acid constituents.16 The leaves of P. macrocarpa contains mangiferin, saponin and polyphenol.17 Phenolics, tannins, flavonoids, alkaloids, and carbohydrates compounds were found in the stem.18 The chemical structures of the vital constituents isolated from P. macrocarpa are shown in Fig. 2.

EXTRACTION OF BIOACTIVE COMPOUNDS Before assessing the potential use of any plant for medicinal purposes, extraction of the bioactive components is a necessary, crucial step. Appropriate selection of the extraction method can establish the exact constituents of interest. Various solvent extraction systems are available for the extraction of phytoconstituents from natural products. The appropriate extraction methods must be considered for the desired components, which can be either


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Bioactive compounds and advanced processing

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Figure 1. Phaleria macrocarpa plant and fruit.

polar or non-polar or both, and its selection also depends on the advantages and disadvantages of the processes. Conventional methods including maceration, percolation, heating under reflux, room temperature solvent extraction and soxhlet extraction are usually used in the extraction of compounds from plants, but have many disadvantages and limitations. Conventional extraction processes are time consuming, e.g. maceration takes up to 7 days, soxhlet 24 h, solvent extraction 2 days where these involve large amounts of organic solvents which are hazardous to health and environment, difficult to remove completely, costly and require high purity of solvent, low extraction selectivity and thermal decomposition of thermo labile compounds. To counter these limitations and setbacks inherent in conventional techniques, more environment-friendly and sophisticated methods that are regarded as non-conventional including solid-phase micro-extraction, supercritical fluid extraction, pressurized-liquid extraction, ultrasound-assisted extraction, microwave-assisted extraction, solid-phase extraction, and surfactant-mediated methods are likely preferences. These methods possess many advantages; in particular the products are free from organic residues. The advantages and disadvantages of conventional and non-conventional methods are listed in Table 1. High extraction yield coupled with effective separation and high concentration of bioactive compounds from a complex plant matrix could be a difficult procedure due to co-extraction of undesirable compounds. Various extraction techniques are used to isolate bioactive compounds (anti diseases) from various plants. The technologically advanced and green methods have innumerable applications because of today’s environmental and health concerns. Numerous studies have been carried out to develop novel extraction processes which are applicable to various compounds. Supercritical fluid extraction (SFE) is an emerging and environmentally safe technology for the extraction of bioactive compounds from natural sources such as plants, food by-products, algae and microalgae. This technique uses nontoxic organic solvents giving reduced pollution, high selectivity, fast extraction, no J Chem Technol Biotechnol (2014)

degradation of active principles and produce products without toxic residues, an attribute sought after in pharmaceutical, food and cosmetic industries.7 SFE mainly depends on certain properties of the fluid such as viscosity, density, diffusivity and dielectric constant, in addition to being able to alter unique operating conditions such as pressure and temperature to reach a supercritical fluid (their solvent power varies over a wide range) and the final product can be easily altered making this technology a good option for the recovery of various types of compounds with high selectivity.19 One of the main interests in SFE is related to the ability to set very precisely their solvent power for different compounds by tuning pressure, temperature and co-solvent content.20 This permits selective fractionation of complex mixtures that cannot be resolved with classical organic solvents or by any other process. SFE process can be applied either for sorting compounds belonging to the same chemical family with different carbon numbers (e.g. fatty acids or oligomers/polymers), or of similar molecular mass but with slightly different polarities. In several cases, it is not possible to avoid the co-extraction of some compound families (with different solubility, but also with different mass transfer resistance within the raw matter). In these cases, it is possible to perform an extraction in successive steps at increasing pressures to obtain fractional extraction of the soluble compounds contained in the organic matrix, selected by decreasing solubility in the supercritical solvent. Fractional separation allows the fractionation of the SFE extracts, operating the plant with some separators in a series at different pressures and temperatures. The commonly used supercritical fluid solvents are ethylene, methane, nitrogen, xenon, fluorocarbons and carbon dioxide. However, carbon dioxide (CO2 ) is used in most of the supercritical fluid separation system because of its safety and low cost. CO2 is non-explosive, nontoxic, and can be easily removed from the final product and possesses the ability to solubilize lipophilic substances, which makes it an attractive alternative to organic solvents. Besides, CO2 is gaseous at room temperature and pressure, which makes compounds recovery very simple resulting in solvent-free extract. However,



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O HO

OMe

HO O

HO OH

O

OH

HO

H O Me

HO

HO HO

O

O OH

OH

O

OH

Figure 2. Chemical structures of constituents isolated from P. macrocarpa.





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Table 1. Advantages and disadvantages of conventional and non conventional extraction method Methods

Advantages

Disadvantages

Solvent extraction: Maceration, percolation, Heat of reflux, Soxhlet, Room temperature solvent extraction

Low processing cost and easy to operate

Pressurized liquid extraction

Faster extraction, lower amount of solvents are used, higher yields are obtained Faster, produces high yields, higher quality of extracts, Not suitable for thermo-labile compounds. lower cost of the extracting agent and environmentally compatible technique Rapid, green technology, provides solvent-free Many parameters to optimize, high investment, extract, low solvent consumption, no filtration difficulty of extracting polar molecules without necessary, high selectivity, nontoxic solvent use, adding modifiers to supercritical fluid. short extraction time (only few mins), cleanliness, safety and simplicity, very good yields, decreased energy consumption and reduced thermal degradation effect

Subcritical fluid extraction

Supercritical fluid extraction

Microwave- assisted extraction

Ultrasound-assisted extraction

Accelerated solvent extraction

CO2 and co-solvent passed through sample

Extraction at desired condition

More effective and selective heating, faster energy transfer, reduced equipment size, faster start-up, and elimination of process steps, environmentally friendly, shorter extraction time (only few min), lower solvent use, moderate investment, rapid, easy to handle, moderate solvent consumption, higher extraction yield, higher selectivity and better quality of target extracts Higher productivity, yield and selectivity, with better processing time and lower solvent consumption, enhanced quality, reduced chemical and physical hazards, and is environmentally friendly, increased mass transfer, better solvent penetration, less dependence on solvent used, extraction at lower temperatures, faster extraction rates and greater yields of product, easy to use, lower investment Rapid, no filtration necessary, low solvent consumption, short extraction time

Sample in an extraction vessel

Extract collected in a collection vessel

CO2 recovered by BPR, remove cosolvent by N2 gas

Figure 3. Flow diagram of supercritical CO2 extraction method.

CO2 has less effect in the extraction of highly polar compounds due to its low polarity. For this reason, co-solvents or modifiers such as hexane, ethanol, methanol, isopropanol, acetonitril, or dichloromethane are used, albeit only in small quantities (less than 15% of CO2 ) for enhancing solubility and selectivity of the extraction.21 Ethanol (food grade) is usually used as a co-solvent due to its nontoxicity and miscibility in CO2 .19 Figure 3 shows the flow diagram of the SFE method. J Chem Technol Biotechnol (2014)

Toxic organic solvents are used, laborious, time consuming (1 to 7 days or more), need large amount of solvent, difficult to remove residual solvent completely, possibility of thermal degradation due to high temperature and lengthy extraction period. Not suitable for thermo-labile compounds.

Extraction solvent must absorb microwave energy, Filtration step required, not suitable when target compounds or solvent are nonpolar, or when the viscosity of solvent is extremely high, not fit for the extraction of thermally labile compounds.

Filtration step required, not suitable for unstable compounds.

Investment high, possible degradation of thermo-labile analytes.

Recently, ultrasound technology has received special attention in the extraction of valuable natural bioactive compounds such as proteins, sugars, polysaccharides-protein complexes, oil, and phenolic compounds from various natural sources.22 Sound waves at more than 20 kHz (up to 100 MHz) pass into the solvent and the better extraction efficiency is related to the acoustic cavitation. When the ultrasound intensity is sufficiently high, the expansion cycle can create cavities or microbubbles in the liquid. Once formed, bubbles will absorb energy from the sound waves and grow during the expansion cycles and recompress during the compression cycle. Bubbles may start another rarefaction cycle or collapse leading to shock waves having extreme conditions of pressure and temperature. Thus, the implosion of cavitation bubbles can hit the surface of the solid matrix and disintegrate the cells causing release of the desired compounds.23 The advantages of this technique over other extraction processes are its simplicity, low equipment cost, high recovery of targeted compounds with low solvent consumption, rapid analysis and better bioactivity properties. On the other hand, the extraction of unstable compounds using ultrasound-assisted extraction (UAE) should be done carefully. The UAE mechanism is given in Fig. 4. Microwave-assisted extraction (MAE) is the most advanced extraction mechanism and has been successfully applied to



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Ultrasound converts Mechanical/ Electrical energy

High frequency vibration

Disruption of cell and release the desired compounds

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Create cavities in the liquid

During implosion, hit the surface of the solid matrix

Expansion of bubble by energy absorption from sound waves

At extreme condition bubble may start collapse

Figure 4. Mechanism of UAE method.

the recovery of natural bioactive compounds such as polyphenols, pigments and polysaccharides from numerous sources.24 Microwaves, a non-contact heat source, are an electromagnetic radiation (wavelength from 0.001 m to 1 m) which can be transmitted as waves. When microwaves pass through a medium, its energy is absorbed and converted into thermal energy from the molecular movements and rotation of liquids with a permanent dipole leading to very fast heating. This principle has been the basis for the improvement of MAE which works by heating the moisture inside the cells and evaporates, producing high pressure on the cell wall. The pressure builds up inside the biomaterial which modifies the physical properties of the biological tissues (cell wall and organelles disrupter) improving the porosity of the biological matrix. This allows better penetration of extracting solvent through the matrix and improved yield of the desired compounds. It heats the matrix internally and externally without a thermal gradient so that natural bioactive compounds can be extracted efficiently and protectively using less energy and solvent volume.23 As a result, this process gives advantages such as higher extraction efficiency, shorter extraction time, reduced energy and solvent consumption, lower environmental pollution and higher level of automation compared with conventional extraction techniques. However, the limitation on the recovery of non-polar compounds with this technique needs to be overcome in industrial applications. The MAE mechanism is described in Fig. 5. Identification of the optimal extraction conditions is one of the main aspects that should be considered in the extraction process. The use of suitable values for different independent variables affecting the extraction could significantly improve the extraction yield of a target component. All these techniques are aimed at replacing toxic and hazardous solvents in many chemical processes in the synthetic laboratory and chemical industry.

BIOLOGICAL ACTIVITY Anti-diabetic activity The enzymes, 𝛼-glucosidase and 𝛼-amylase are present in the brush border of the small intestine and in the pancreas, respectively, which are responsible for the digestion of carbohydrates. The enzyme converts oligosaccharides, disaccharides and starch to glucose and other monosaccharide. Inhibition of these enzymes reduces the rate of carbohydrate digestion, thus reducing the breakdown of carbohydrates into glucose. Glucose absorption, therefore, reduces and blood glucose level decreases contributing to the hypoglycemic effect. Phaleria macrocarpa fruit extracts have profound anti-diabetic activity.18,25 The n-butanol extract of young and ripened fruits followed by ethyl acetate extract and


then methanol extract has the highest anti-diabetic activity. Phaleria macrocarpa fruit extract inhibits 𝛼-amylase and 𝛼-glucosidase delaying glucose absorption and lowering postprandial hyperglycemia. The presence of carbohydrate compounds may be responsible for this activity which is thought to be a competitive inhibitor of 𝛼-glucosidase.18 Methanol extracts of pericarp is recently reported to decrease blood glucose due to the presence of magniferin in the most active n-butanol sub-fraction of methanol.26 Anti-oxidant activity The antioxidant activity of an extract is associated with its free radical scavenging activity. Different types of assays have been developed to determine the antioxidant properties of plant extracts, such as the ferric thiocyanate assay, thiobarbituric acid assay, ferric reducing antioxidant power assay and DPPH (2,2-diphenyl-1-picryl-hydroxyl) assay.1,6 DPPH is a free radical used to determine antioxidant properties of plant extracts and is a strong indicator for determining antioxidant capacity in human plasma.5 Flavonoids have most effective antioxidant properties and less toxicity compared with synthetic antioxidants. Hendra et al.1 reported the presence of kaempferol, myricetin, naringin, quercetin, and rutin as major flavonoids present in P. macrocarpa fruit. Fruits and leaves of P. macrocarpa are rich in flavanoids and phenolics which make it a potent antioxidant.6 Oskoueian et al.27 reported the role of phenolic compounds (methanolic extracts of Jatropha curcas Linn) in antioxidant activity and their ability to act as free radical and nitric oxide (NO) scavengers, leading to the formation of phenoxyl radicals. The phenol constituents such as magniferin, gallic acid and 6-dihydroxy-4-methoxybenzophenone-2-O-𝛽-D-glucoside present in leaves, mesocarp, pericarp and seed extract of P. macrocarpa are responsible for antioxidant activity.28 Anti-hypercholesterolemic activity Hypercholesterolemia is caused when total cholesterol level is increased in the blood owing to obesity, food habit, sedentary life style, smoking and sometimes genetic abnormalities. These factors increase low-density lipoproteins (LDL), i.e. bad cholesterol, and reduce the levels of high-density lipoproteins (HDL), i.e. good cholesterol. Many diseases such as atherosclerosis, heart disease, stroke and hypertension are caused by cholesterol elevation. High cholesterol level in the cells inhibits the LDL-R (low-density lipoproteins receptor) and pro-protein converters subtilisin/kexin type-9 (PCSK-9) transcription. As a result, the cells can intake less plasma cholesterol, and cholesterol level in blood becomes high. Gallic acid present in P. macrocarpa reduces cholesterol level in the body by increasing LDL-R and PCSK-9.12 It enhances the




Microwave

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Heating moisture inside the cell and evaporates

Converted into thermal energy

Release the desired compound

Allow better penetration of extracting solven through the matrix

Producing high pressure on the cell wall

Improving(create) porosity of the biological matrix

Figure 5. Mechanism of MAE method.

binding of LDL particles in the blood to LDL-R and causes regulation of cell surface LDL-R, which thus decreases cholesterol levels.5 Anti-hypertensive activity The P. macrocarpa plant has been used traditionally to treat many diseases, such as hypertension, heart diseases and so on.11,12 The anti-hypertension and cure for heart diseases are found in the leaves, fruits and seeds of P. macrocarpa.29,30 The kaempferol (flavanoid) has an important effect on the cardiovascular system that reduces the risk of heart diseases.5 Icariside isolated from chloroform extracts of P. macrocarpa fruit have a moderate vasorelaxant property which reduces high blood pressure. It mainly enhances the vasorelaxant responses of isoproterenol and inhibits noradrenaline induced contractions contributing to the increase of second messengers such as cyclic adenosine mono phosphate and cyclic guanosine mono phosphate by inhibition of phosphodiesterase and activation of adenylatecyclase.14 Anti-microbial activity Antibacterial activities have been shown in leaves and seeds of P. macrocarpa.16 Phaleria macrocarpa contain flavanoids, saponins, polyphenols and tannins that have greater inhibition activity against gram positive bacteria than gram-negative bacteria.31 These bacteria are Bacillus cereus, Bacillus subtilis, Enterobacter aerogenes, Eschericia coli, Klebsiella pneumonia, Micrococcus luteus, Pseudomonas aeroginosa and Staphylococcus aureus. The bioactive compounds exhibit their antimicrobial activity by different mechanisms such as inhibiting nucleic acid synthesis and energy metabolism or cytoplasmic membrane function. Kaempferols are found to inhibit Staphylococcus aureus, Enterococcus faecalis, Escherichi coli and Pseudomonas aeroginosa. The methanol extracts of P. macrocarpa have shown good activity against Pseudomonas aeroginosa and strong activity against Escherichia coli, Bacillus cereus and Streptococcus aureus.5 The ethyl acetate extract has also shown strong activity against Pseudomonas aeroginosa, Streptococcus aureus and Bacillus cereus and good activity against Escherichia coli, Klebsiella pneumoniae and Streptococcus ubellis. The lowest activity has been found in n-hexane and chloroform extract. The seed of P. macrocarpa contains phorbolesters which inhibits the growth of certain fungi such as Aspergillus niger, Fusarium oxysporum, Ganoderma lucidum and Mucor indicus.2 Anti-inflammatory activity Inducible nitric oxide synthase (iNOS) enzyme produces nitric oxide (NO) which creates inflammation. Therefore, inhibition of nitric oxide production is the aim in anti-inflammatory treatment. Fruits and leaves of P. macrocarpa are found to have strong anti-inflammatory activity. Moderate anti-inflammatory J Chem Technol Biotechnol (2014)

activity has been found in pericarp and mesocarp extracts of fruit and weak activity in seed reported by Hendra et al.1 Constituents including tannins, terpenoids, flavanoids, saponins and polyphenols might be responsible for anti-inflammation. The anti-inflammation activity has been found in semipolar methanolic extract containing 20.26% phalerin (DLBS1425) of P. macrocarpa fruit.32 Recent work has found that phalerin has mild inhibitory effects on xanthine oxidase and lipo-oxygenase while its inhibitory effect on hyaluronidase was found to be non-significant.33 A proprietary and standardized semi polar bioactive extract DLBS1442 of P. macrocarpa fruit is preclinical-proven to have anti-inflammatory activity. DLBS1442 is effective in alleviating primary dysmenorrheal, abdominal pain and other symptoms related to premenstrual syndrome. The clinical study has shown that DLBS1442 (benzophenone glycoside) is safe and well tolerated for dysmenorrheal and/or premenstrual.13 Anti-carcinogenic activity From leaves to root, every part of P. macrocarpa plants (leaves, bark, stem, seed and fruit) is being used as a traditional medicine for different types of cancer especially against breast cancer 34,35 and brain tumor.36 Phaleria macrocarpa is also used as a supplement with adriamycin cyclophosphamide (AC) for reducing tumor growth in breast cells by inducing apoptosis and at the same time, it protects liver and kidney from damage caused by AC.37 Phalerin and gallic acid has a major contribution in its cytotoxic properties.11,38 Gallic acid from fruits of P. macrocarpa selectively induces cancer cell death in various cancer cells, such as human esophageal cancer, gastric cancer, colon cancer, breast cancer, cervix cancer, and malignant brain tumor.28 Methanolic semipolar extract (DLBS1425) of P. macrocarpa containing phalerin was proven to exert its anticancer activity in breast cancer cells by acting as an anti-proliferative, anti-angiogenic and apoptotic inducer.32 Leaves extract of P. macrocarpa has mild toxicity to HepG2 cell lines, and the presence of phenolic compounds in the leaves extract of P. macrocarpa may reduce the cell number since reactive oxygen has an important role in carcinogenesis.6 Besides, ethanolic leaves extracts of P. macrocarpa is also reported to produce antitumor activity.5 Anti-infertility (male) activity Infertility is one of the most serious problems around the world. According to the United States Food and Drug Administration (FDA), infertility can be caused by androgen deficiency or low testosterone level. Phaleria macrocarpa has been shown to have the potential to increase secretion of testosterone hormone in the presence of saponin.39 Phaleria macrocarpa can be an alternative medication to improve male fertility by improving sperm quality. Table 2 shows the phytoconstituents isolated from P. macrocarpa with their respective biological activities.



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Table 2. Phytoconstituents isolated from P. macrocarpa with their respective biological activity P. macrocarpa Leaves, stem, seed and fruits Pericarp Fruits Leaves, seed, fruits Fruits

Bark, leaves, mesocarp, pericarp and seed Fruits, leaves and seed Fruits Fruit, seed Whole plant

Bioactive compounds

References

Phalerin, gallic acid Phalerin Gallic acid Flavanoids, saponins, polyphenols, tannins Terpenoids, saponins, tannins, flavanoids and phenols such as rutin and cathecol, phalerin, benzophenone glucoside Flavanoids, phenolics,gallic acid, 6, hydroxyl-4-methoxy-benzophenone -2-O-𝛽-D-glucoside Flavonoids(Kaempferol), icaricide Saponin

Anticancer Antidiabetic Antihyperlipidemic Antibacterial and antifungal Anti inflammatory

Des-acetylfevicordin A, and its derivatives Mahkoside A, dodecanoic acid, palmitic acid, desacetyl flavicordin A, flavicordin A, flavicordin D, flavicordin A glucoside, ethyl stearate, lignans, alkaloids, saponins, sucrose

Toxicity Anti-microbial

TOXICITY STUDIES The extracts of P. macrocarpa have a number of valuable medicinal properties as claimed traditionally and in scientific works. In spite of this, the plant is also recognized for its poisonous consequence. Unprocessed ripened fruit of P. macrocarpa cause oral ulcers (consumed as traditional medicine). However, the constituents of P. macrocarpa fruits responsible for this effect have not been identified and quantified so far.6 Eating P. macrocarpa is reported to trigger embryo-fetotoxicity in female mice.40 Butanol extracts of ripened fruits is reported to cause mild necrosis of proximal convoluted tubules in mice kidney and ethanol extracts of P. macrocarpa are reported to cause mild hepatic hypertrophy and an increase in serum glutamate pyruvate transaminase in Javanese Quail.5 Like fruits, seeds of P. macrocarpa have also shown their toxicity: des-acetylfevicordin-A and its derivatives isolated from the seeds of P. macrocarpa are reported to exert toxicity in brine shrimp (Artemiasalina).41

RECENT STUDIES ON THE RECOVERY OF NATURAL BIOACTIVE COMPOUNDS FROM P. MACROCARPA USING CONVENTIONAL AND NON-CONVENTIONAL EXTRACTION METHODS There is no report on the use of non-conventional extraction methods to extract bioactive compounds from P. macrocarpa except extraction of magniferin using subcritical water extraction4 and supercritical carbon dioxide extraction of highly unsaturated oil from P. macrocarpa seed.42 Subcritical water extraction (SWE) was performed at a temperature of 50–150 ∘ C; pressure of 0.7 to 4.0 MPa and extraction time of 1 to 7 h. At optimal extraction conditions of 373 K, 4.0 MPa and extraction time 5 h, the extraction yield of mangiferin was 21.7 mg g−1 . This value was close to the extraction yield with methanol (25.0 mg g−1 ) and higher than those with water (18.6 mg g−1 ) or ethanol (13.2 mg g−1 ) at their boiling points. In the SFE method, response surface methodology (RSM) with central composite design (CCD) was employed to determine


Biological activity

Antioxidant

Vasorelaxant Increase male fertility

1,11,28,32,34,35,37,38 18,25,26 12 2,6,31,44 1,13,33

1,6,25,34

11,12,14,35 39 41 2

the best combination of variables to obtain high extraction yield in the supercritical CO2 process. Three parameters, temperature, pressure, and flow rate of CO2 were considered as independent variables. The variables and their levels were temperature, 60 to 80 ∘ C; pressure, 25 to 45 MPa CO2 flow rate, 3 to 5 mL min−1 . Palmitic acid, linoleic acid, oleic acid, stearic acid, gondoic acid were found. The optimum conditions obtained from RSM were 72 ∘ C temperature, 42 MPa pressure and 4.5 mL min−1 CO2 flow rate. Several conventional methods that have been used in the extraction of bioactive compounds from P. macrocarpa are discussed below. The extraction of seed oil from P. macrocarpa seed was carried out by solvent extraction method using n-hexane under optimal conditions. The optimal conditions were 72 ∘ C temperatures, 8.4 h extraction time and 10.9 mL g−1 solvent-to-feed ratio for seed oil extraction. The main chemical constituents of the oil, determined by GC–MS and FTIR, were oleic acid and linoleic acid as the main fatty acid constituents.16 A maceration method has been used to extract benzophenone glucoside (2,4’-dihydroxy-4-methoxy-benzophenone-6-O-𝛽-Dglucopyranoside) compound from the bark of P. macrocarpa. In this method, n-hexane, ethyl acetate and ethanol have been used as solvents. HPLC were used to separate benzophenone glucoside, which has inhibitory activity against leukemia L1210 cell line.35 Susilawati et al.8 also used a maceration method to extract benzophenone (2,6,4’-trihydroxy-4-methoxybenzophenone) from the dried leaves of P. macrocarpa. Methanol was used as the solvent and the extraction time was 24 h. Benzophenone has antioxidant activity due to the phenolic group.8 The fruits of P. macrocarpa were extracted by maceration using methanol solvent. The methanol extract was partitioned between ethyl acetate and water to give an active ethyl acetate extract and then was subjected to chromatography giving two active fractions. The active fractions were combined and then further subjected to chromatography to yield three active fractions. Further purification of the active fraction yielded an active compound of gallic acid, which has significant inhibition of cell proliferation in a series




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of cancer cell lines and induced apoptosis in esophageal cancer cells.28 Phalerin had been extracted using maceration with methanol on P. macrocarpa leaves that had been dried and ground at room temperature for 24 h. The extracted phalerin was verified before use in an anti-inflammatory test and was found to be cytotoxic to myeloma cell line.15,33,43 Hot water extract of P. macrocarpa fruits can significantly increase sperm viability without changing sperm motility and morphology. Hence, P. macrocarpa can be used as an alternative to improve male fertility by improving the sperm quality. Saponin may be responsible for this activity because it has the potency to increase testosterone hormone level, which is the main male reproductive hormone and this hormone plays a major role in sperm quality.39 The extraction of bioactive compounds from P. macrocarpa has also been performed by a maceration and percolation method.13 P. macrocarpa was macerated with methanol for 15 min and percolated for 45 min at 50 ∘ C. The extract contained bezophenone glucoside (DLBS1442) which has been found to be able to alleviate discomfort linked to premenstrual syndrome and primary dysmenorrheal. Extractions of phenolic and flavonoid compounds from different parts (pericarp, mesocarp and seed) of P. macrocarpa fruit using reflux at 90 ∘ C using methanol followed by HCl resulted in total phenolic contents in mesocarp, pericarp and seed extract of 60.5, 59.2 and 47.7 mg galic acid equivalent per gram of dry weight, respectively. The total flavonoid content in mesocarp, pericarp, and seed extract was 161.3 131.7 ± 1.66 and 35.9 ± 2.47 mg rutin equivalent per gram of dry weight, respectively. Good antioxidant and anti-inflammatory activities have been shown in pericarp and mesocarp extracts due to the presence of phenolic and flavonoid compounds.1 Dried and ground P. macrocarpa powder (fruits and leaves) was sequentially extracted with petroleum ether and then methanol using a Soxhlet apparatus (40 ∘ C) for 48 h. The methanol extract was further fractionated to obtain chloroform, ethyl acetate, n-butanol and aqueous fractions, which were tested for antidiabetic activity. Further fractionation of n-butanol fraction yielded sub-fractions I and II. Phytochemical screening showed the presence of flavonoids, terpenes and tannins in methanol extract, n-butanol extract and sub-fraction I extract. LC-MS analysis revealed the presence of mangiferin, which may be responsible for antidiabetic activity at 9.52%, 33.30% and 22.50% in methanol extract, n-butanol extract and sub-fraction I extract, respectively.26 Stems of P. macrocarpa were extracted by maceration with methanol and further fractionated with water–ethyl acetate. The water fraction was refractionated by n-butanol. Among ethyl acetate, n-butanol and water fraction extracts the ethyl acetate fraction extracts had the highest inhibition activity (antidiabetic). Phenolic, tannins, flavonoids, alkaloids and carbohydrates have been found in the stems and the activity may be due to the carbohydrate compound.18 Shodikin44 also carried out the extraction of bioactive compounds from dried and ground leaves of P. macrocarpa by maceration for 20 h using ethanol. Extract from the leaves contained flavonoids, polyphenols, saponins and tannins compounds that have antimicrobial activity. Sliced and air-dried P. macrocarpa fruits were boiled, filtered and freeze-dried and used in controlling the body weight of obese people and for treating hypercholesterolemia. The aqueous extract enhances low-density lipoprotein receptor and pro-protein converters subtilisin/kexin type-9 J Chem Technol Biotechnol (2014)

expression. The anti-hypercholesterolemic property may be due to the gallic acid compound found in P. macrocarpa fruit.12 The dried leaves of P. macrocarpa were extracted five times at room temperature successively by n-hexane, chloroform, ethyl acetate and methanol. The extracts were tested for antibacterial, antioxidant and cytotoxic properties which have been linked to the presence of polyphenolic compounds.6 The dried and ground fruits of P. macrocarpa were macerated with chloroform for 24 h at room temperature, and then the residue was macerated for 24 h with methanol. The chloroform extract was tested by chromatography and then the fourth fraction was purified by HPLC to give icariside C3 (sesquiterpene glucoside). The first fraction was purified by HPLC to give 2,4’,6-trihydroxy-4-methoxybenzophenone-2-O-𝛽-D-glucoside. The methanol extract was subject to chromatography and then the fraction eluted from 60% methanol was purified by HPLC to give 2,4’,6-trihydroxy-4 methoxybenzo phenone-2-O-𝛽-D-glucoside and mangiferin.14 The dried fruits of P. macrocarpa were extracted for 48 h using ethanol at room temperature. Then ethanol crude extract was tested using chromatography by chloroform and petroleum ether, which gave benzophenone (1) and benzophenone (2). From the literature and spectroscopic data analysis, the structures of benzophenone were deduced as (1) 2,6,4’-trihydroxy-4-methoxybenzophenone and (2) 6,4’-dihydroxy -4-methoxybenzo-phenone-2-O-𝛽-D-glucopyranoside, respectively, the former having a weak cytotoxic effect and the latter being nontoxic.45 From a literature review, it was seen that all the methods used were conventional and used huge amounts of organic solvent. They are toxic to humans and dangerous for the environment. So, the aim of this review was to find environment-friendly, less labour intensive, low cost, fast, better biologically active compounds extraction by non-conventional process. The modern and most convenient methods such as supercritical fluid extraction (SFE) ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) can be used for the extraction of bioactive compound from P. macrocarpa. Table 3 shows extraction of P. macrocarpa using conventional extraction method.

CONCLUSION This review compared various conventional and non-conventional extraction methods for separating bioactive compounds from P. macrocarpa. Plant extracts contain a large variety of bioactive compounds with many other constituents including pharmaceuticals and nutraceuticals that require separations, purifications and fractionations for further processing. Pharmaceutical and nutraceutical industries are always looking for green processing methods obtaining pure products although processing methods especially for the extraction, purification and isolation are still limited to conventional methods. Non-conventional methods such as SFE, MAE, UAE, and SWE are of great interest owing to their efficiency (fast, organic-residues-free, low temperature processing and cost effective). Moreover, non-conventional methods are technologically advanced and claimed to be green with innumerable applications. There is a dearth of reports on non-conventional extraction of P. macrocarpa. Bioactive compounds of P. macrocarpa extracted by non-conventional methods may retain the natural quality free of organic residues that can



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MT.S Easmin et al.

Table 3. Extraction of P. macrocarpa using conventional extraction method P. macrocarpa Bark (2,4′ -dihydroxy-4-methoxybenzophenone-6-O-𝛽-Dglucopyranoside) Fruit (gallic acid) Leaves (phalerin) Fruit (benzophenone glucoside) Seed (Oleic acid, Linoleic acid) Leaves (benzophenone) Leaves (flavonoids, polyphenols, saponins and tannins) Pericarp, mesocarp and seed (phenolics, flavanoids) Fruit Fruit Stem (phenolics, tannins, flavonoids, alkaloids, carbohydrates) Fruit (gallic acid) Leaves (polyphenolic compound) Fruit (icaricideC3 , phalerin, magniferin) Fruit (benzophenone)

Extraction method

Reference

Maceration

Ethanol, n-hexane, ethyl acetate

Maceration Maceration Maceration then percolation Solvent extraction method Maceration Maceration

Methanol, ethyl acetate. water Methanol Methanol n-hexane Methanol Ethanol

Heat of reflux

Methanol

1

Soxhlet, then maceration

Petrol ether, methanol, water

46

Boiled in water then centrifuged Maceration

water Methanol, ethyl acetate, n- butanol, water

39 18

Boiled with water Room temperature solvent extraction Maceration Room temperature solvent extraction

water n-hexane, chloroform, ethyl acetate, methanol Chloroform, methanol Ethanol

12 6 14 45

be further exploited for the pharmaceutical, cosmetic, functional foods and neutraceuticals industries. The environmental issues and health concerns of society are also demanding green technology.

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