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Bioactivecompoundsandadvancedprocessingtechnology:Phaleriamacrocarpa(sheff.)Boerl,areview

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Md.MoklesurRahmanSarker

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ReviewReceived: 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 advancedprocessing technology: Phaleria macrocarpa(sheff.) Boerl, a reviewMst. Sabina Easmin,a Md. Zaidul Islam Sarker,a* Sahena Ferdosh,b SitiHadijah Shamsudin,a Kamaruzzaman Bin Yunus,b Md. Salim Uddin,a Md.Moklesur Rahman Sarker,c Md. Jahurul Haque Akanda,d Md. SohrabHossaine and HPS Abdul Khalile

Abstract

Recent technological advances and the development of new methods has provided an opportunity to obtain highly purifiednatural bioactive compound extracts with potential for the treatment and prevention of human diseases. The use of hazardousand toxic solvents for the extraction and processing of bioactive compounds from plant materials is considered a problemfor health, safety and environmental pollution. Advanced technology aims to increase production of the desired compoundsand find an alternative to using toxic solvents in the extraction of bioactive compounds from plant materials. The evergrowing interest in plant bioactive compounds and today’s concerns about environment issues have led to an increasedneed for an efficient and green extraction method. This review is focused on the extraction of bioactive compounds fromplants 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 updatedoverview of the bioactive compounds present in the plant Phaleria macrocarpa and its extraction, fractionation, purificationand isolation is provided. The advantages and disadvantages of both conventional and non-conventional extraction methodsare also discussed in this review.© 2014 Society of Chemical Industry

Keywords: medicinal plant; Phaleria macrocarpa; extraction of bioactive compounds; conventional extraction method; non-conven-tional extraction method

INTRODUCTIONAt 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 bioac-tive compounds gives an excellent pool of molecules that pro-duce essential functional foods, neutraceuticals, food additivesand pharmaceuticals for human health benefits. Extracts frommedicinal plants provide huge opportunities for new drug dis-coveries because of the structural complexity and unprofitabilityof chemical synthesis. Also, synthetic drugs have been associatedwith many side effects on human health.2 Environment-friendlyand organic-residues-free extraction methods, termed green tech-nology, for the extraction of bioactive compounds from medicinalplants are currently a most interesting research area. The major-ity of the extraction solvents are organic chemicals that are haz-ardous and toxic to health and environment. This is exacerbatedby the high cost and ensuing environmental problems from thelarge amount of waste by-products from these chemical industries.Organic solvents produce greenhouse gases in the environmentthat are dangerous for humans, agriculture, microorganisms, etc.Of late, a mounting interest has developed for a safer and greener

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

∗ Correspondence to: Md. Zaidul Sarker, Faculty of Pharmacy, InternationalIslamic University Malaysia, Kuantan Campus, 25200 Kuantan, Pahang,Malaysia. E-mail: zaidul@iium.edu.my or zaidulsarker@yahoo.com

a Faculty of Pharmacy, International Islamic University Malaysia, Kuantan Cam-pus, 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, 50603Kuala Lumpur, Malaysia

d Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSIUniversity, 56000 Kuala Lumpur, Malaysia

e School of Industrial Technology, Universiti Sains Malaysia, 11800 Pulau Pinang,Malaysia

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solvents are not needed. Limitations of conventional methods aswell as the necessity for pure products in food, cosmetics, neu-traceuticals and pharmaceuticals encourage efficient, green andalternative extraction methods. Green extraction methods couldbe defined as extraction processes that reduce energy consump-tion, allow use of alternative solvents or without solvents, anduse of renewable natural sources ensuring a safe and high qualityproduct. The non-conventional methods are considered as ‘greenprocessing’. These methods are able to either cut down or reducethe consumption of organic solvents and degradation of samples,elimination of valuable materials of the sample, clean-up and con-centration steps before chromatographic analysis, improvement inextraction efficiency, selectivity and/or kinetics of the extraction.The ease of automation of these techniques also favors their usefor the extraction of plant materials.3

More than 80% of the world’s population depends on tradi-tional medicine in treating ailments, as reported by the WorldHealth Organization (WHO).3 Plants contain a wide range of com-pounds promising as traditional medicine to treat chronic as wellas infectious diseases. Thousands of phytochemicals from plantshave been considered safe and effective alternatives with fewerside effects. These phytochemicals, which are secondary metabo-lites of plants, include tannins, terpenoids, alkaloids, flavonoidsand others, which have many biological remedial benefits such asanticancer, antimicrobial, antioxidant, antidiarrheal, analgesic andwound healing. These bioactive compounds are used to developeither drugs or dietary supplements.1

Phaleria macrocarpa (Scheff.) Boerl is a popular medicinal plantin many south Asian countries. Traditionally, P. macrocarpa isused to control cancer, impotency, hemorrhoids, diabetes mellitus,allergies, liver and heart diseases, kidney disorders, blood relateddiseases, acne, stroke, migraine, and various skin ailments.1,4 Everypart of this plant from leaves to roots is used for the prevention ofdiseases with some good results. The stems, leaves and fruits of P.macrocarpa have been widely used for medicinal treatments. Theconstituents of the stem are used to treat bone cancer; seeds areused in treating breast cancer, cervicle cancer, lung, liver and heartdiseases; while the leaves are used to treat impotence, diabetesmellitus, allergies, tumors and blood diseases.5 This herb has beenused in both unprocessed and processed form as tea, juice andother liquid forms. The unprocessed fruit may have toxicity andsometime are poisonous.6

Due to the side effects of synthetic drugs and the develop-ment of microbial resistance to chemically synthesized drugs,researchers are interested in the extraction, isolation and char-acterization of bioactive compounds from natural sources. Thegrowing awareness of the health benefits has led to an interestin consuming foods enriched with bioactive compounds. Theimportant steps needed to utilize bioactive compounds from nat-ural sources are extraction, pharmacological screening, isolationand characterization, and toxicological and clinical evaluation.3

However, the toxicity of the solvents, the degradation of thecompounds, consumption of time, total yield and selectivity ofthe process are major setbacks in the extraction process. Thesepoints are directly linked to environment pollution, economicviability, quality and purity of the final product.7 The limitationsof conventional methods as well as the demand for organicresidue-free products (green products) in food, cosmetics, neu-traceuticals and pharmaceuticals highlight the need for efficient,green and alternative (non-conventional) extraction methods. Inthis review, chemical constituents, biological activities and theextraction of bioactive compounds from P. macrocarpa have been

extensively discussed. From published reports it can be seen thatextraction from this plant was carried out using only conventionalextraction methods. The extraction of bioactive compounds fromP. macrocarpa using non-conventional methods could provide itsapplication in food, cosmetic, and pharmaceutical industries toobtain products free from organic residues.

Phaleria macrocarpa and its chemical constituentsPhaleria macrocarpa (Scheff.) Boerl (Thymelaceae family) is com-monly known as Mahkota Dewa, pau or God’s crown. This plantis 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 growsthroughout the year in tropical areas, reaching a height ofbetween 1 and 6 m. It is a complete tree consisting of stem, leaves,flower and fruit, the fruit being elliptical with a diameter of around3 cm. The color of the fruit is green before ripening and turnsred 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, ethylstearate, lignans and sucrose.9 Mahkoside-A (4,4′ dihydroxy-2-methoxybenzophenone-6-O-𝛽-D-glucopyranoside) was firstisolated from the pit of P. macrocarpa along with six other knowncompounds 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 partsof P. macrocarpa by chiral column analysis.11 Saponins, alka-loids, polyphenolics, phenols, flavanoids, lignans and tanninsare found in the bark and fruit.12,13 Fruit contains icarisideC3, magniferin, and gallic acid.2,11,14 Phalerin was first iso-lated 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 proposedstructure (2,4,6-trihydroxy-4-methoxy-benzophenone-3-O-𝛽-D-glucoside) 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 peri-carp of the fruit and the mesocarp and seed contain naringinand quercitin.2 Phorboesters, des-acetyl flavicordin-A and29-norcucurbitacin derivatives have been isolated from seed. Themain chemical constituents of the oil extracted from seed wereoleic acid and linoleic acid as the main fatty acid constituents.16

The leaves of P. macrocarpa contains mangiferin, saponin andpolyphenol.17 Phenolics, tannins, flavonoids, alkaloids, and car-bohydrates compounds were found in the stem.18 The chemicalstructures of the vital constituents isolated from P. macrocarpa areshown in Fig. 2.

EXTRACTION OF BIOACTIVE COMPOUNDSBefore assessing the potential use of any plant for medicinalpurposes, extraction of the bioactive components is a necessary,crucial step. Appropriate selection of the extraction method canestablish the exact constituents of interest. Various solvent extrac-tion systems are available for the extraction of phytoconstituentsfrom natural products. The appropriate extraction methods mustbe considered for the desired components, which can be either

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Figure 1. Phaleria macrocarpa plant and fruit.

polar or non-polar or both, and its selection also depends on theadvantages and disadvantages of the processes. Conventionalmethods including maceration, percolation, heating under reflux,room temperature solvent extraction and soxhlet extraction areusually used in the extraction of compounds from plants, buthave many disadvantages and limitations. Conventional extrac-tion processes are time consuming, e.g. maceration takes upto 7 days, soxhlet 24 h, solvent extraction 2 days where theseinvolve large amounts of organic solvents which are hazardousto health and environment, difficult to remove completely, costlyand require high purity of solvent, low extraction selectivity andthermal decomposition of thermo labile compounds. To counterthese limitations and setbacks inherent in conventional tech-niques, more environment-friendly and sophisticated methodsthat are regarded as non-conventional including solid-phasemicro-extraction, supercritical fluid extraction, pressurized-liquidextraction, ultrasound-assisted extraction, microwave-assistedextraction, solid-phase extraction, and surfactant-mediatedmethods are likely preferences. These methods possess manyadvantages; in particular the products are free from organicresidues.

The advantages and disadvantages of conventional andnon-conventional methods are listed in Table 1.

High extraction yield coupled with effective separation andhigh concentration of bioactive compounds from a complex plantmatrix could be a difficult procedure due to co-extraction of unde-sirable compounds. Various extraction techniques are used to iso-late bioactive compounds (anti diseases) from various plants. Thetechnologically advanced and green methods have innumerableapplications because of today’s environmental and health con-cerns. Numerous studies have been carried out to develop novelextraction processes which are applicable to various compounds.

Supercritical fluid extraction (SFE) is an emerging and environ-mentally safe technology for the extraction of bioactive com-pounds from natural sources such as plants, food by-products,algae and microalgae. This technique uses nontoxic organic sol-vents giving reduced pollution, high selectivity, fast extraction, no

degradation of active principles and produce products withouttoxic residues, an attribute sought after in pharmaceutical, foodand cosmetic industries.7 SFE mainly depends on certain proper-ties of the fluid such as viscosity, density, diffusivity and dielec-tric constant, in addition to being able to alter unique operatingconditions such as pressure and temperature to reach a supercrit-ical fluid (their solvent power varies over a wide range) and thefinal product can be easily altered making this technology a goodoption for the recovery of various types of compounds with highselectivity.19

One of the main interests in SFE is related to the ability to setvery precisely their solvent power for different compounds bytuning pressure, temperature and co-solvent content.20 This per-mits selective fractionation of complex mixtures that cannot beresolved with classical organic solvents or by any other process.SFE process can be applied either for sorting compounds belong-ing to the same chemical family with different carbon numbers(e.g. fatty acids or oligomers/polymers), or of similar molecularmass but with slightly different polarities. In several cases, it is notpossible to avoid the co-extraction of some compound families(with different solubility, but also with different mass transferresistance within the raw matter). In these cases, it is possible toperform an extraction in successive steps at increasing pressuresto obtain fractional extraction of the soluble compounds con-tained in the organic matrix, selected by decreasing solubility inthe supercritical solvent. Fractional separation allows the fraction-ation of the SFE extracts, operating the plant with some separatorsin a series at different pressures and temperatures. The commonlyused supercritical fluid solvents are ethylene, methane, nitrogen,xenon, fluorocarbons and carbon dioxide. However, carbon diox-ide (CO2) is used in most of the supercritical fluid separation systembecause of its safety and low cost. CO2 is non-explosive, nontoxic,and can be easily removed from the final product and possessesthe ability to solubilize lipophilic substances, which makes it anattractive alternative to organic solvents. Besides, CO2 is gaseousat room temperature and pressure, which makes compoundsrecovery very simple resulting in solvent-free extract. However,

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HO

HO

HO

OH

OHOO

O Me

H

O

OH

HO

HO

HO

O

O

O

OHOH

HO

OMe

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 temperaturesolvent extraction

Low processing cost and easy to operate Toxic organic solvents are used, laborious, timeconsuming (1 to 7 days or more), need largeamount of solvent, difficult to remove residualsolvent completely, possibility of thermaldegradation due to high temperature and lengthyextraction period.

Pressurized liquid extraction Faster extraction, lower amount of solvents are used,higher yields are obtained

Not suitable for thermo-labile compounds.

Subcritical fluid extraction Faster, produces high yields, higher quality of extracts,lower cost of the extracting agent andenvironmentally compatible technique

Not suitable for thermo-labile compounds.

Supercritical fluid extraction Rapid, green technology, provides solvent-freeextract, low solvent consumption, no filtrationnecessary, high selectivity, nontoxic solvent use,short extraction time (only few mins), cleanliness,safety and simplicity, very good yields, decreasedenergy consumption and reduced thermaldegradation effect

Many parameters to optimize, high investment,difficulty of extracting polar molecules withoutadding modifiers to supercritical fluid.

Microwave- assisted extraction More effective and selective heating, faster energytransfer, reduced equipment size, faster start-up,and elimination of process steps, environmentallyfriendly, shorter extraction time (only few min),lower solvent use, moderate investment, rapid, easyto handle, moderate solvent consumption, higherextraction yield, higher selectivity and betterquality of target extracts

Extraction solvent must absorb microwave energy,Filtration step required, not suitable when targetcompounds or solvent are nonpolar, or when theviscosity of solvent is extremely high, not fit for theextraction of thermally labile compounds.

Ultrasound-assisted extraction Higher productivity, yield and selectivity, with betterprocessing time and lower solvent consumption,enhanced quality, reduced chemical and physicalhazards, and is environmentally friendly, increasedmass transfer, better solvent penetration, lessdependence on solvent used, extraction at lowertemperatures, faster extraction rates and greateryields of product, easy to use, lower investment

Filtration step required, not suitable for unstablecompounds.

Accelerated solvent extraction Rapid, no filtration necessary, low solventconsumption, short extraction time

Investment high, possible degradation ofthermo-labile analytes.

Extraction at desired condition

CO2 and co-solvent passed through sample

CO2 recovered by BPR, remove co-solvent by N2 gas

Extract collected in a collectionvessel

Sample in an extraction vessel

Figure 3. Flow diagram of supercritical CO2 extraction method.

CO2 has less effect in the extraction of highly polar compoundsdue to its low polarity. For this reason, co-solvents or modifierssuch as hexane, ethanol, methanol, isopropanol, acetonitril, ordichloromethane are used, albeit only in small quantities (lessthan 15% of CO2) for enhancing solubility and selectivity of theextraction.21 Ethanol (food grade) is usually used as a co-solventdue to its nontoxicity and miscibility in CO2.19 Figure 3 shows theflow diagram of the SFE method.

Recently, ultrasound technology has received special attentionin the extraction of valuable natural bioactive compounds suchas proteins, sugars, polysaccharides-protein complexes, oil, andphenolic compounds from various natural sources.22 Sound wavesat more than 20 kHz (up to 100 MHz) pass into the solvent andthe better extraction efficiency is related to the acoustic cavita-tion. When the ultrasound intensity is sufficiently high, the expan-sion cycle can create cavities or microbubbles in the liquid. Onceformed, bubbles will absorb energy from the sound waves andgrow during the expansion cycles and recompress during thecompression cycle. Bubbles may start another rarefaction cycleor collapse leading to shock waves having extreme conditions ofpressure and temperature. Thus, the implosion of cavitation bub-bles can hit the surface of the solid matrix and disintegrate thecells causing release of the desired compounds.23 The advantagesof this technique over other extraction processes are its simplic-ity, low equipment cost, high recovery of targeted compoundswith low solvent consumption, rapid analysis and better bioac-tivity properties. On the other hand, the extraction of unstablecompounds using ultrasound-assisted extraction (UAE) should bedone carefully. The UAE mechanism is given in Fig. 4.

Microwave-assisted extraction (MAE) is the most advancedextraction mechanism and has been successfully applied to

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Ultrasound converts Mechanical/ Electrical

energy

Disruption of cell and release the desired

compounds

High frequency vibration

During implosion, hitthe surface of the

solid matrix

Create cavities in the liquid

At extreme condition bubble may start

Expansion of bubble by energy absorption from

sound waves

collapse

Figure 4. Mechanism of UAE method.

the recovery of natural bioactive compounds such as polyphe-nols, pigments and polysaccharides from numerous sources.24

Microwaves, a non-contact heat source, are an electromagneticradiation (wavelength from 0.001 m to 1 m) which can be trans-mitted as waves. When microwaves pass through a medium, itsenergy is absorbed and converted into thermal energy from themolecular movements and rotation of liquids with a permanentdipole leading to very fast heating. This principle has been thebasis for the improvement of MAE which works by heating themoisture inside the cells and evaporates, producing high pressureon the cell wall. The pressure builds up inside the biomaterialwhich modifies the physical properties of the biological tissues(cell wall and organelles disrupter) improving the porosity of thebiological matrix. This allows better penetration of extractingsolvent through the matrix and improved yield of the desiredcompounds. It heats the matrix internally and externally withouta thermal gradient so that natural bioactive compounds canbe extracted efficiently and protectively using less energy andsolvent volume.23 As a result, this process gives advantages suchas higher extraction efficiency, shorter extraction time, reducedenergy and solvent consumption, lower environmental pollutionand higher level of automation compared with conventionalextraction techniques. However, the limitation on the recovery ofnon-polar compounds with this technique needs to be overcomein industrial applications. The MAE mechanism is described inFig. 5.

Identification of the optimal extraction conditions is one of themain aspects that should be considered in the extraction process.The use of suitable values for different independent variablesaffecting the extraction could significantly improve the extractionyield of a target component.

All these techniques are aimed at replacing toxic and hazardoussolvents in many chemical processes in the synthetic laboratoryand chemical industry.

BIOLOGICAL ACTIVITYAnti-diabetic activityThe enzymes, 𝛼-glucosidase and 𝛼-amylase are present in thebrush border of the small intestine and in the pancreas, respec-tively, which are responsible for the digestion of carbohydrates.The enzyme converts oligosaccharides, disaccharides and starchto glucose and other monosaccharide. Inhibition of these enzymesreduces the rate of carbohydrate digestion, thus reducing thebreakdown of carbohydrates into glucose. Glucose absorption,therefore, reduces and blood glucose level decreases contribut-ing to the hypoglycemic effect. Phaleria macrocarpa fruit extractshave profound anti-diabetic activity.18,25 The n-butanol extract ofyoung and ripened fruits followed by ethyl acetate extract and

then methanol extract has the highest anti-diabetic activity. Pha-leria macrocarpa fruit extract inhibits 𝛼-amylase and 𝛼-glucosidasedelaying glucose absorption and lowering postprandial hyper-glycemia. The presence of carbohydrate compounds may beresponsible for this activity which is thought to be a compet-itive inhibitor of 𝛼-glucosidase.18 Methanol extracts of pericarpis recently reported to decrease blood glucose due to the pres-ence of magniferin in the most active n-butanol sub-fraction ofmethanol.26

Anti-oxidant activityThe antioxidant activity of an extract is associated with its freeradical scavenging activity. Different types of assays have beendeveloped to determine the antioxidant properties of plantextracts, such as the ferric thiocyanate assay, thiobarbituricacid assay, ferric reducing antioxidant power assay and DPPH(2,2-diphenyl-1-picryl-hydroxyl) assay.1,6 DPPH is a free radicalused to determine antioxidant properties of plant extracts and isa strong indicator for determining antioxidant capacity in humanplasma.5 Flavonoids have most effective antioxidant propertiesand less toxicity compared with synthetic antioxidants. Hendraet al.1 reported the presence of kaempferol, myricetin, naringin,quercetin, and rutin as major flavonoids present in P. macrocarpafruit.

Fruits and leaves of P. macrocarpa are rich in flavanoids andphenolics which make it a potent antioxidant.6 Oskoueianet al.27 reported the role of phenolic compounds (methanolicextracts of Jatropha curcas Linn) in antioxidant activity andtheir ability to act as free radical and nitric oxide (NO) scav-engers, leading to the formation of phenoxyl radicals. Thephenol constituents such as magniferin, gallic acid and6-dihydroxy-4-methoxybenzophenone-2-O-𝛽-D-glucosidepresent in leaves, mesocarp, pericarp and seed extract of P.macrocarpa are responsible for antioxidant activity.28

Anti-hypercholesterolemic activityHypercholesterolemia is caused when total cholesterol level isincreased in the blood owing to obesity, food habit, sedentarylife style, smoking and sometimes genetic abnormalities. Thesefactors increase low-density lipoproteins (LDL), i.e. bad choles-terol, and reduce the levels of high-density lipoproteins (HDL), i.e.good cholesterol. Many diseases such as atherosclerosis, heart dis-ease, stroke and hypertension are caused by cholesterol elevation.High cholesterol level in the cells inhibits the LDL-R (low-densitylipoproteins receptor) and pro-protein converters subtilisin/kexintype-9 (PCSK-9) transcription. As a result, the cells can intakeless plasma cholesterol, and cholesterol level in blood becomeshigh. Gallic acid present in P. macrocarpa reduces cholesterol levelin the body by increasing LDL-R and PCSK-9.12 It enhances the

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Microwave

Release the desiredcompound

Converted intothermal energy

Allow better penetration ofextracting solven through the

matrix

Heating moistureinside the cell and

evaporates

Improving(create)porosity of the

biological matrix

Producing highpressure on the cell

wall

Figure 5. Mechanism of MAE method.

binding of LDL particles in the blood to LDL-R and causes reg-ulation of cell surface LDL-R, which thus decreases cholesterollevels.5

Anti-hypertensive activityThe P. macrocarpa plant has been used traditionally to treat manydiseases, such as hypertension, heart diseases and so on.11,12 Theanti-hypertension and cure for heart diseases are found in theleaves, fruits and seeds of P. macrocarpa.29,30 The kaempferol (fla-vanoid) has an important effect on the cardiovascular system thatreduces the risk of heart diseases.5 Icariside isolated from chloro-form extracts of P. macrocarpa fruit have a moderate vasorelaxantproperty which reduces high blood pressure. It mainly enhancesthe vasorelaxant responses of isoproterenol and inhibits nora-drenaline induced contractions contributing to the increase of sec-ond messengers such as cyclic adenosine mono phosphate andcyclic guanosine mono phosphate by inhibition of phosphodi-esterase and activation of adenylatecyclase.14

Anti-microbial activityAntibacterial activities have been shown in leaves and seeds of P.macrocarpa.16 Phaleria macrocarpa contain flavanoids, saponins,polyphenols and tannins that have greater inhibition activityagainst gram positive bacteria than gram-negative bacteria.31

These bacteria are Bacillus cereus, Bacillus subtilis, Enterobacteraerogenes, Eschericia coli, Klebsiella pneumonia, Micrococcus luteus,Pseudomonas aeroginosa and Staphylococcus aureus. The bioac-tive compounds exhibit their antimicrobial activity by differentmechanisms such as inhibiting nucleic acid synthesis and energymetabolism or cytoplasmic membrane function. Kaempferolsare found to inhibit Staphylococcus aureus, Enterococcus fae-calis, Escherichi coli and Pseudomonas aeroginosa. The methanolextracts of P. macrocarpa have shown good activity against Pseu-domonas aeroginosa and strong activity against Escherichia coli,Bacillus cereus and Streptococcus aureus.5 The ethyl acetate extracthas also shown strong activity against Pseudomonas aeroginosa,Streptococcus aureus and Bacillus cereus and good activity againstEscherichia coli, Klebsiella pneumoniae and Streptococcus ubellis.The lowest activity has been found in n-hexane and chloroformextract. The seed of P. macrocarpa contains phorbolesters whichinhibits the growth of certain fungi such as Aspergillus niger,Fusarium oxysporum, Ganoderma lucidum and Mucor indicus.2

Anti-inflammatory activityInducible nitric oxide synthase (iNOS) enzyme produces nitricoxide (NO) which creates inflammation. Therefore, inhibition ofnitric oxide production is the aim in anti-inflammatory treat-ment. Fruits and leaves of P. macrocarpa are found to havestrong anti-inflammatory activity. Moderate anti-inflammatory

activity has been found in pericarp and mesocarp extracts offruit and weak activity in seed reported by Hendra et al.1 Con-stituents including tannins, terpenoids, flavanoids, saponinsand polyphenols might be responsible for anti-inflammation.The anti-inflammation activity has been found in semipolarmethanolic extract containing 20.26% phalerin (DLBS1425)of P. macrocarpa fruit.32 Recent work has found that pha-lerin has mild inhibitory effects on xanthine oxidase andlipo-oxygenase while its inhibitory effect on hyaluronidasewas found to be non-significant.33 A proprietary and standardizedsemi polar bioactive extract DLBS1442 of P. macrocarpa fruit ispreclinical-proven to have anti-inflammatory activity. DLBS1442is effective in alleviating primary dysmenorrheal, abdominal painand other symptoms related to premenstrual syndrome. The clini-cal study has shown that DLBS1442 (benzophenone glycoside) issafe and well tolerated for dysmenorrheal and/or premenstrual.13

Anti-carcinogenic activityFrom leaves to root, every part of P. macrocarpa plants (leaves,bark, stem, seed and fruit) is being used as a traditional medicinefor different types of cancer especially against breast cancer 34,35

and brain tumor.36 Phaleria macrocarpa is also used as a sup-plement with adriamycin cyclophosphamide (AC) for reducingtumor growth in breast cells by inducing apoptosis and at thesame time, it protects liver and kidney from damage caused byAC.37 Phalerin and gallic acid has a major contribution in its cyto-toxic properties.11,38 Gallic acid from fruits of P. macrocarpa selec-tively induces cancer cell death in various cancer cells, such ashuman esophageal cancer, gastric cancer, colon cancer, breastcancer, cervix cancer, and malignant brain tumor.28 Methanolicsemipolar extract (DLBS1425) of P. macrocarpa containing pha-lerin was proven to exert its anticancer activity in breast cancercells by acting as an anti-proliferative, anti-angiogenic and apop-totic inducer.32 Leaves extract of P. macrocarpa has mild toxicity toHepG2 cell lines, and the presence of phenolic compounds in theleaves extract of P. macrocarpa may reduce the cell number sincereactive oxygen has an important role in carcinogenesis.6 Besides,ethanolic leaves extracts of P. macrocarpa is also reported to pro-duce antitumor activity.5

Anti-infertility (male) activityInfertility 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 lowtestosterone level. Phaleria macrocarpa has been shown to havethe potential to increase secretion of testosterone hormone in thepresence of saponin.39 Phaleria macrocarpa can be an alternativemedication to improve male fertility by improving sperm quality.

Table 2 shows the phytoconstituents isolated from P. macrocarpawith their respective biological activities.

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Table 2. Phytoconstituents isolated from P. macrocarpa with their respective biological activity

P. macrocarpa Bioactive compounds Biological activity References

Leaves, stem, seed and fruits Phalerin, gallic acid Anticancer 1,11,28,32,34,35,37,38Pericarp Phalerin Antidiabetic 18,25,26Fruits Gallic acid Antihyperlipidemic 12Leaves, seed, fruits Flavanoids, saponins, polyphenols, tannins Antibacterial and antifungal 2,6,31,44Fruits Terpenoids, saponins, tannins, flavanoids and

phenols such as rutin and cathecol, phalerin,benzophenone glucoside

Anti inflammatory 1,13,33

Bark, leaves, mesocarp,pericarp and seed

Flavanoids, phenolics,gallic acid, 6,hydroxyl-4-methoxy-benzophenone-2-O-𝛽-D-glucoside

Antioxidant 1,6,25,34

Fruits, leaves and seed Flavonoids(Kaempferol), icaricide Vasorelaxant 11,12,14,35Fruits Saponin Increase male fertility 39

Fruit, seed Des-acetylfevicordin A, and its derivatives Toxicity 41Whole plant Mahkoside A, dodecanoic acid, palmitic acid,

desacetyl flavicordin A, flavicordin A,flavicordin D, flavicordin A glucoside, ethylstearate, lignans, alkaloids, saponins, sucrose

Anti-microbial 2

TOXICITY STUDIESThe extracts of P. macrocarpa have a number of valuable medicinalproperties as claimed traditionally and in scientific works. In spiteof this, the plant is also recognized for its poisonous consequence.Unprocessed ripened fruit of P. macrocarpa cause oral ulcers (con-sumed as traditional medicine). However, the constituents of P.macrocarpa fruits responsible for this effect have not been iden-tified and quantified so far.6 Eating P. macrocarpa is reported totrigger embryo-fetotoxicity in female mice.40 Butanol extracts ofripened fruits is reported to cause mild necrosis of proximal con-voluted tubules in mice kidney and ethanol extracts of P. macro-carpa are reported to cause mild hepatic hypertrophy and anincrease in serum glutamate pyruvate transaminase in JavaneseQuail.5 Like fruits, seeds of P. macrocarpa have also shown their tox-icity: des-acetylfevicordin-A and its derivatives isolated from theseeds of P. macrocarpa are reported to exert toxicity in brine shrimp(Artemiasalina).41

RECENT STUDIES ON THE RECOVERYOF NATURAL BIOACTIVE COMPOUNDS FROMP. MACROCARPA USING CONVENTIONALAND NON-CONVENTIONAL EXTRACTIONMETHODSThere is no report on the use of non-conventional extractionmethods to extract bioactive compounds from P. macrocarpaexcept extraction of magniferin using subcritical water extraction4

and supercritical carbon dioxide extraction of highly unsaturatedoil from P. macrocarpa seed.42 Subcritical water extraction (SWE)was performed at a temperature of 50–150 ∘C; pressure of 0.7to 4.0 MPa and extraction time of 1 to 7 h. At optimal extractionconditions of 373 K, 4.0 MPa and extraction time 5 h, the extractionyield of mangiferin was 21.7 mg g−1. This value was close to theextraction yield with methanol (25.0 mg g−1) and higher thanthose with water (18.6 mg g−1) or ethanol (13.2 mg g−1) at theirboiling points.

In the SFE method, response surface methodology (RSM) withcentral composite design (CCD) was employed to determine

the best combination of variables to obtain high extractionyield in the supercritical CO2 process. Three parameters, tem-perature, pressure, and flow rate of CO2 were considered asindependent variables. The variables and their levels were tem-perature, 60 to 80 ∘C; pressure, 25 to 45 MPa CO2 flow rate, 3 to5 mL min−1. Palmitic acid, linoleic acid, oleic acid, stearic acid,gondoic acid were found. The optimum conditions obtained fromRSM 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 extrac-

tion of bioactive compounds from P. macrocarpa are discussedbelow.

The extraction of seed oil from P. macrocarpa seed was carriedout by solvent extraction method using n-hexane under optimalconditions. The optimal conditions were 72 ∘C temperatures, 8.4 hextraction time and 10.9 mL g−1 solvent-to-feed ratio for seed oilextraction. The main chemical constituents of the oil, determinedby GC–MS and FTIR, were oleic acid and linoleic acid as the mainfatty acid constituents.16

A maceration method has been used to extract benzophenoneglucoside (2,4’-dihydroxy-4-methoxy-benzophenone-6-O-𝛽-D-glucopyranoside) compound from the bark of P. macrocarpa. Inthis method, n-hexane, ethyl acetate and ethanol have been usedas 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 extractbenzophenone (2,6,4’-trihydroxy-4-methoxybenzophenone) fromthe dried leaves of P. macrocarpa. Methanol was used as the solventand the extraction time was 24 h. Benzophenone has antioxidantactivity due to the phenolic group.8

The fruits of P. macrocarpa were extracted by maceration usingmethanol solvent. The methanol extract was partitioned betweenethyl acetate and water to give an active ethyl acetate extract andthen was subjected to chromatography giving two active fractions.The active fractions were combined and then further subjectedto chromatography to yield three active fractions. Further purifi-cation of the active fraction yielded an active compound of gallicacid, which has significant inhibition of cell proliferation in a series

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of cancer cell lines and induced apoptosis in esophageal cancercells.28

Phalerin had been extracted using maceration with methanolon P. macrocarpa leaves that had been dried and ground at roomtemperature for 24 h. The extracted phalerin was verified beforeuse in an anti-inflammatory test and was found to be cytotoxic tomyeloma cell line.15,33,43

Hot water extract of P. macrocarpa fruits can significantlyincrease sperm viability without changing sperm motility andmorphology. Hence, P. macrocarpa can be used as an alternativeto improve male fertility by improving the sperm quality. Saponinmay be responsible for this activity because it has the potencyto increase testosterone hormone level, which is the main malereproductive hormone and this hormone plays a major role insperm quality.39

The extraction of bioactive compounds from P. macrocarpa hasalso been performed by a maceration and percolation method.13

P. macrocarpa was macerated with methanol for 15 min and per-colated for 45 min at 50 ∘C. The extract contained bezophenoneglucoside (DLBS1442) which has been found to be able to allevi-ate discomfort linked to premenstrual syndrome and primary dys-menorrheal.

Extractions of phenolic and flavonoid compounds from differentparts (pericarp, mesocarp and seed) of P. macrocarpa fruit usingreflux at 90 ∘C using methanol followed by HCl resulted in totalphenolic 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 rutinequivalent per gram of dry weight, respectively. Good antioxidantand anti-inflammatory activities have been shown in pericarp andmesocarp extracts due to the presence of phenolic and flavonoidcompounds.1

Dried and ground P. macrocarpa powder (fruits and leaves) wassequentially extracted with petroleum ether and then methanolusing a Soxhlet apparatus (40 ∘C) for 48 h. The methanol extractwas further fractionated to obtain chloroform, ethyl acetate,n-butanol and aqueous fractions, which were tested for antidia-betic activity. Further fractionation of n-butanol fraction yieldedsub-fractions I and II. Phytochemical screening showed the pres-ence of flavonoids, terpenes and tannins in methanol extract,n-butanol extract and sub-fraction I extract. LC-MS analysisrevealed the presence of mangiferin, which may be responsiblefor antidiabetic activity at 9.52%, 33.30% and 22.50% in methanolextract, n-butanol extract and sub-fraction I extract, respectively.26

Stems of P. macrocarpa were extracted by maceration withmethanol and further fractionated with water–ethyl acetate. Thewater fraction was refractionated by n-butanol. Among ethylacetate, n-butanol and water fraction extracts the ethyl acetatefraction extracts had the highest inhibition activity (antidiabetic).Phenolic, tannins, flavonoids, alkaloids and carbohydrates havebeen found in the stems and the activity may be due to the car-bohydrate compound.18

Shodikin44 also carried out the extraction of bioactive com-pounds from dried and ground leaves of P. macrocarpa by mac-eration for 20 h using ethanol. Extract from the leaves containedflavonoids, polyphenols, saponins and tannins compounds thathave antimicrobial activity. Sliced and air-dried P. macrocarpa fruitswere boiled, filtered and freeze-dried and used in controllingthe body weight of obese people and for treating hypercholes-terolemia. The aqueous extract enhances low-density lipopro-tein receptor and pro-protein converters subtilisin/kexin type-9

expression. The anti-hypercholesterolemic property may be due tothe gallic acid compound found in P. macrocarpa fruit.12

The dried leaves of P. macrocarpa were extracted five timesat room temperature successively by n-hexane, chloroform,ethyl acetate and methanol. The extracts were tested forantibacterial, antioxidant and cytotoxic properties which havebeen linked to the presence of polyphenolic compounds.6

The dried and ground fruits of P. macrocarpa were maceratedwith chloroform for 24 h at room temperature, and then theresidue was macerated for 24 h with methanol. The chloroformextract was tested by chromatography and then the fourthfraction was purified by HPLC to give icariside C3 (sesquiter-pene glucoside). The first fraction was purified by HPLC to give2,4’,6-trihydroxy-4-methoxybenzophenone-2-O-𝛽-D-glucoside.The methanol extract was subject to chromatography andthen the fraction eluted from 60% methanol was purifiedby HPLC to give 2,4’,6-trihydroxy-4 methoxybenzophenone-2-O-𝛽-D-glucoside and mangiferin.14

The dried fruits of P. macrocarpa were extracted for 48 husing ethanol at room temperature. Then ethanol crudeextract was tested using chromatography by chloroform andpetroleum ether, which gave benzophenone (1) and ben-zophenone (2). From the literature and spectroscopic dataanalysis, 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, respec-tively, the former having a weak cytotoxic effect and the latterbeing nontoxic.45

From a literature review, it was seen that all the methods usedwere 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, lesslabour intensive, low cost, fast, better biologically active com-pounds extraction by non-conventional process. The modern andmost convenient methods such as supercritical fluid extraction(SFE) ultrasound-assisted extraction (UAE) and microwave-assistedextraction (MAE) can be used for the extraction of bioactive com-pound from P. macrocarpa.

Table 3 shows extraction of P. macrocarpa using conventionalextraction method.

CONCLUSIONThis review compared various conventional and non-conventionalextraction methods for separating bioactive compounds from P.macrocarpa. Plant extracts contain a large variety of bioactivecompounds with many other constituents including pharmaceu-ticals and nutraceuticals that require separations, purificationsand fractionations for further processing. Pharmaceutical andnutraceutical industries are always looking for green processingmethods obtaining pure products although processing methodsespecially for the extraction, purification and isolation are stilllimited to conventional methods. Non-conventional methodssuch as SFE, MAE, UAE, and SWE are of great interest owingto their efficiency (fast, organic-residues-free, low temperatureprocessing and cost effective). Moreover, non-conventionalmethods are technologically advanced and claimed to be greenwith innumerable applications. There is a dearth of reports onnon-conventional extraction of P. macrocarpa. Bioactive com-pounds of P. macrocarpa extracted by non-conventional methodsmay retain the natural quality free of organic residues that can

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www.soci.org MT.S Easmin et al.

Table 3. Extraction of P. macrocarpa using conventional extraction method

P. macrocarpa Extraction method Solvent Reference

Bark (2,4′-dihydroxy-4-methoxy-benzophenone-6-O-𝛽-D-glucopyranoside)

Maceration Ethanol, n-hexane, ethyl acetate 35

Fruit (gallic acid) Maceration Methanol, ethyl acetate. water 28Leaves (phalerin) Maceration Methanol 15,33,43Fruit (benzophenone glucoside) Maceration then percolation Methanol 13Seed (Oleic acid, Linoleic acid) Solvent extraction method n-hexane 16Leaves (benzophenone) Maceration Methanol 8Leaves (flavonoids, polyphenols, saponins

and tannins)Maceration Ethanol 44

Pericarp, mesocarp and seed (phenolics,flavanoids)

Heat of reflux Methanol 1

Fruit Soxhlet, then maceration Petrol ether, methanol, water 46

Fruit Boiled in water then centrifuged water 39Stem (phenolics, tannins, flavonoids,

alkaloids, carbohydrates)Maceration Methanol, ethyl acetate, n- butanol, water 18

Fruit (gallic acid) Boiled with water water 12Leaves (polyphenolic compound) Room temperature solvent extraction n-hexane, chloroform, ethyl acetate, methanol 6Fruit (icaricideC3, phalerin, magniferin) Maceration Chloroform, methanol 14Fruit (benzophenone) Room temperature solvent extraction Ethanol 45

be further exploited for the pharmaceutical, cosmetic, func-tional foods and neutraceuticals industries. The environmentalissues and health concerns of society are also demanding greentechnology.

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