The Extraction of Bioactive Agents from Calophyllum inophyllum L., and Their Pharmacological Properties

: Calophyllum inophyllum L. has been used for many generations by Pacific Islanders because of its numerous health and aesthetic advantages. The leaves, stems, roots, fruits, flowers, and seeds of this plant contain significant phytochemicals, including flavonoids, coumarins, fatty acids, and xanthones, which have been shown to have wound healing, analgesic, anti-inflammatory, antiaging, anti-arthritic, anti-cancer, anti-proliferative, anti-diabetic, anti-microbial, and anti-HIV effects. The chemical profiles and bioactive potential may vary due to different extraction techniques, plant parts, and geographical origins. Extraction is the essential first step in the analysis of bioactive compounds that leads to further separation, identification, and characterization. Conventional methods like maceration, Soxhlet, and percolation are often used to extract bioactive compounds from C. inophyllum . However, little study has been carried out on non-conventional methods such as pressured liquid extraction, supercritical fluid extraction (SFE), and ultrasound-assisted extraction. The SFE method can be used to extract bioactive compounds from C. inophyllum to retain their pharmacological properties for application in pharmaceutical and cosmetic products.


Introduction
Calophyllum inophyllum L. (Calophyllaceae), locally called "Daok" in Guam, is a woody pantropical plant widely distributed in Asia, Africa, and the Pacific regions.It is also known as Tamanu, Beauty leaf, Alexandrian laurel, Indian laurel, Beach Calophyllum, Beach touriga, Kamani, Laurelwood, Honne, etc., in different regions of the world.Daok is a slow-growing medium-to-large tree, up to 20 m in height, with shiny leathery leaves, furrowed bark, fragrant white flowers, spherical brown fruits with large seeds, and a broad spreading crown of irregular branches [1].In Guam, it grows wildly on shores and forests, and is often planted as an ornamental plant.This tree produces fragrant gums that exuded from its trunk and bark, as well as strong, quality wood that the natives of Guam used to make big canoes, carabao cartwheels, and boat timber [1].Pacific Islanders have traditionally used different parts of this plant, such as nuts, leaves, roots, bark, and fruits, for their various health and beauty benefits [2].The oil extracted from C. inophyllum nuts contains resinous neoflavanoid and pyranocoumarin compounds beside common fatty acids, which are known to have different medicinal properties [2].
Bioactive compounds in plants are secondary metabolites having pharmacological or toxicological effects on both humans and animals.The main chemical groups of bioactive compounds in plants are alkaloids, glycosides, flavonoids, and proanthocyanidins; tannins, mono-, di-, and sesqui-terpenoids; phenylpropanoids, resins, furocoumarins, and naphthodianthrones; and proteins and peptides [3].The predominant classes of phytoconstituents present in the genus Calophyllum are coumarins, xanthones, chromanones, triterpenes, steroids, and glycosides [4].Plant bioactive compounds are receiving increased attention due to their huge demand in food, pharmaceutical, and nutraceutical industries.
Extraction is the most crucial initial step that plays an important role in the final result and outcome of analyses for the phytochemicals study.Several conventional and non-conventional methods are employed for the extraction of bioactive compounds.For instance, maceration, Soxhlet extraction, percolation, hydro distillation, and reflux are commonly used as conventional methods, whereas ultrasound-assisted extraction, enzymeassisted extraction, microwave-assisted extraction, and supercritical fluid extraction are considered non-conventional or modern techniques [5].Many studies have discussed the extraction, isolation, and characterization of bioactive compounds from different parts of C. inophyllum [2,4].Until now, no single method was regarded as the standard for extracting bioactive compounds from C. inophyllum.
The present review focuses on conventional and non-conventional extraction techniques and factors that affect the extraction efficiency of bioactive compounds from C. inophyllum.Ethnobotanical uses, major bioactive compounds, and their pharmacological properties are also discussed.

Ethnobotany and Ethnomedicinal Uses
C. inophyllum seed oil has been utilized as a traditional medicine to treat skin healing and wound care, pain relief, anti-insect and insect repellent, oral health, joint pain, scar reduction, anti-aging and skincare, hair and scalp care, earaches, and respiratory issues by many cultures throughout its native Southeast Asia and Pacific Islands for generations.In French Polynesia, seed oil has been traditionally used for topical applications on the skin, as well as mucous membrane lesions to cure all kinds of dermal affections (burns, dermatoses, eczema, acne, psoriasis, chilblains, skin cracks, diabetic sores, hemorrhoids, dry skin, etc.) [2].These trees were considered sacred and planted inside sacred sanctuaries before the Polynesians converted to Christianity.Léguillier et al. [6] confirmed the wound healing and antimicrobial properties of the seed oil collected from Indonesia, Tahiti, Fiji Islands, and New Caledonia, which has been traditionally used topically to treat a wide range of skin injuries [6].In India, C. inophyllum's roots are used in a decoction to cure ulcers, boils, and ophthalmia, the bark is used to treat orchitis, the latex is applied topically to treat rheumatism and psoriasis, and the leaves are used in a decoction to treat eye infections [4].In Thai traditional medicine, the mangrove plant C. inophyllum is used to treat various ailments such as wound healing, arthritis, and skin diseases [7].In Madagascar, C. inophyllum seeds are used to treat scabies and other skin diseases, and to alleviate the inflammation in case of dental caries [8].In Vietnam, C. inophyllum seed oil has been used as a traditional medicine to treat burns, skin-related and rheumatic diseases, and insomnia [9].
According to Friday and Ogoshi [10], the wood of this plant has been traditionally used for canoe and boat building by the Pacific islanders, while today it is used for cabinet making and furniture, as well as turning and carving.They also reported that leaves have been applied topically to treat wounds and skin diseases, while soaking in water was used to treat eye issues.Seed oil has been used for centuries to treat skin injuries and as a cosmetic such as massage oil and skin moisturizer [10].Dweck and Meadows [11] reported detailed traditional uses of C. inophyllum in Africa, Asia, Polynesia, and the Pacific Islands.For example, (1) the tree is thought to have diuretic properties in Java, but in Samoa, the entire plant is considered toxic; the milky juice can cause blindness and an injection of its sap into the bloodstream is said to result in death; (2) the gum extracted from wounded barks has been used to cure wounds and ulcers; (3) the bark has been used to treat chronic bronchitis, phthisis, internal hemorrhages, and gonorrhea; (4) the decoction of the root has been applied topically to heal ulcers and treat heatstroke; (5) native black communities in Papua New Guinea have traditionally utilized charred leaves as a treatment for acne, rashes, boils, and wounds.These leaves are used by the indigenous people of Samoa and New Caledonia to heal wounds, leg ulcers, and inflammations of the skin.The leaves are applied to irritated eyes by skimming the oily surface of water that has been soaked with leaves in Madagascar, India, and Fiji.(6) Dark-skinned Fijians highly valued the seed oil for treating bruises, arthritis, and joint discomfort.In most South Sea islands, tamanu oil is utilized as a wound and ulcer remedy [11].In Guam, nut oil is used for external medicine for an assortment of skin issues, sap from the trunk and bark is used as glue for tools and canoe making, and wood is used for the keel of the canoe.

Major Bioactive Compounds of C. inophyllum and Their Therapeutic Effects
The major bioactive compounds and their reported biological activities from different parts (fruits, seeds, leaf, stem, root, and flower) of C. inophyllum are presented in Table 1.The main bioactive compounds of French Polynesian "tamanu oil" resinous part from the seed are calophyllolide, inopyllums (C, D, E, P), calanolides (A, B, D), and tamanolides [2].According to Léguillier et al. [6], bioactive fatty acids from the seed oil exhibited accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control, and distinct antibacterial activity against Staphylococcus aureus strain involved in skin infections.The authors also reported the lowest and highest cytotoxicity value at LC 50 18.7% and 7.3%, respectively.The bioactive compound (4-(3-methylazetidin-1-yl) pentan-2-ol) of C. inophyllum seeds oil demonstrated potent antibacterial activities (17 ± 1.73 to 24 ± 1.15 mm) against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa [12].Nguyen et al. [9] isolated the bioactive calophyllolide compound from seeds and reported their anti-inflammatory and wound-healing activities.Based on their results, calophyllolide reduced fibrosis formation and effectively promoted wound closure in mouse models without causing body weight loss.The "Tamanu oil emulsion" demonstrated evidence of wound healing activity, collagen and glycosaminoglycan synthesis, and cell proliferation in experiments performed on human skin cells (keratinocytes and dermal fibroblasts) [13].These biological activities could be facilitated by the bioactive neoflavanoid's molecule present in Tamanu oil emulsion [13].According to research carried out by Ku et al. [14], C. inophyllum seed oils showed the highest UVA (wavelength 320-400 nm) and UVB (wavelength 290-320 nm) absorption efficiencies, making them a potential source of high-efficiency natural screening agents.The results showed maximum inhibition at 250 µg/mL in proteinase inhibition and hemolysis assays, and the LD 50 of stem and seed extract was found to be greater than 5000 and 2000 mg/kg/p.o., respectively.One novel chromanone acid derivative, namely inocalophylline C, was identified from the nut oil resin of C. inophyllum L., widely distributed in Vietnam [15].Table 1.Bioactive compounds and their pharmacological properties from different parts of C. inophyllum.
According to Navyasri et al. [22], fractions 6 and 7 of the leaf extract's bioactive constituents showed moderate-to-strong anti-inflammatory and anti-arthritis activity, confirming the presence of flavonoids, tannins, and phenolic compounds.Various pyranocoumarins, calophyllolide, inophyllums B, C, G1, G2, and P were determined from 136 leaf extracts of C. inophyllum of French Polynesia [23].The results confirmed the geographical distribution of inophyllums and indicate those rich in HIV-1 active (+)-inophyllums [23].
Young fruit extracts of C. inophyllum exhibited antiaging properties via upregulating superoxide dismutase and Sirtuin 1 genes, and reduced oxidative stress in cerevisiae BY611 [26].The presence of phenolic compounds in fruit peel were responsible for the antibacterial activity against S.aureus and Mycobacterium smegmatis [30].Shanmugapriya et al. [37] found cytotoxic effects against MCF-7 cells in a dose-dependent manner, with the IC 50 for 24 h of 23.59 µg/mL.

Factors Affecting the Extraction Efficiency of Bioactive Compounds
There are several factors that affect the extraction efficiency of bioactive components from plants.The major influencing factors include the characteristic of the sample matrix, type of solvents, sample-to-solvent ratio, extraction time and temperature, pressure, and extraction technique.Bioactive compounds are varied not only with the different species of Calophyllum, but also different parts of the plant such as the leaves, stem bark, roots, fruit kernel, and heartwood [38].The selection of solvent polarity is the most significant factor that affects the mass transfer, as well as the solubility of bioactive compounds.Rajendran and Gurunathan [39] examined the ultrasonic-assisted heterogeneous solvent technique to extract seed oil from C. inophyllum and found that the equal ratio of diethyl ether and ethanol in assistance with ultrasonic waves yields maximum oil extraction.They also reported the optimal conditions, which included a seed kernel-to-solvent ratio of 1:15 (w:v), 45 min, 36 • C temperature, and 54% ultrasonication power, yielding a maximum bio-oil yield of 82.8%.(w/w).The effect of solvent polarity levels to separate the bioactive compounds (xanthone and coumarin) from C. inophyllum leaf extract were performed using methanol (polar solvent) and hexane (non-polar solvent) with the solvent ratio of 1 [40].According to the authors, 50% methanol gives the best performance whereby coumarin and xanthones were separated in the methanol fraction (81.18% recovery) and the hexane fraction (81.91% recovery), respectively [40].Different extraction methods (conventional and ultrasonic), ethanol concentrations (0%, 30%, 50%, 70%, or 95%), and processing temperatures (drying and freezing) of five Bulgarian medicinal plants were studied to investigate their effects on bioactive compounds [41].According to the authors, the yield content from dried samples with 70% ethanol using the conventional method was nearly twice that of the ultrasonic extraction method [41].
Temperature and time were reported to affect the extraction efficiency of bioactive compounds from C. inophyllum.The total phenolic and flavonoid compounds of C. inophyllum leaves were significantly affected by solvent types, the concentration of solvent in water, length of extraction time, and temperature [24].The optimal conditions for the extraction of the coumarin mixture were 90% ethanol and eight stages of extraction, which contributed to 92.95 ± 3.76% of recovery with the highest DPPH inhibition (57.72 ± 2.70%), with an IC50 value of 305 ppm [24].The total phenol and flavonoid content of aqueous extracts of Humulus lupulus L. was found to increase with increasing temperature from 25 • C to 57.5 • C [42].However, the concentration of bioactive compounds decreased at temperatures of ≥90 • C due to their denaturation at high temperatures and exposure time [42].The relative importance of every factor influencing extraction efficiency primarily depends on the particular technique employed.For example, choosing the right solvent is crucial for maceration, but temperature, pressure, solvent concentration, and flow rate are also crucial for supercritical fluid extraction.The essential factors influencing microwave-assisted extraction include the solvent-to-feed ratio, plant sample matrix, extraction temperature and time, solvent composition, microwave power, and energy density [43].

Extraction Technique for Bioactive Compounds
Extraction is the most crucial step in the isolation and purification of bioactive agents from plant tissue.The choice of extraction technique depends on various factors, including the target bioactive compounds of interest, process efficiency, production cost, product quality, available equipment, and environmental impact [44][45][46].Extraction can be conducted using conventional and non-conventional techniques, each offering distinct advantages based on the desired compounds and the application.The advantages and disadvantages of using conventional and nonconventional methods for the extraction of bioactive compounds from different plants are described by many authors [5,[44][45][46].

Conventional Extraction Technique
Several conventional extraction techniques have been employed to extract bioactive compounds from C. inophyllum using various solvents, including maceration, percolation, Soxhlet, and dynamic maceration.The common extraction techniques with the extraction conditions and solvents are briefly described in Table 2. Seed oil [51] Maceration is the most common conventional methods used for the extraction of bioactive compounds from C. inophyllum.Mai et al. [47] investigated the maceration extraction process of seeds oil and their antioxidant and antimicrobial activity.The highest oil extraction was obtained at a material size of 2 mm, moisture content of 4%, acetone solvent with a materials/solvent (w/v) ratio of 1:30, extraction time of 4 h, and extraction temperature of 50 • C [47].Total phenolic, flavonoid, and saponin contents were obtained from C. inophyllum flower via maceration with sequential extraction using hexane, ethyl acetate, and methanol [7].The highest yield (58.89%) was obtained with methanol; as the polarity of ethyl acetate and hexane decreased, the yields dropped to 5.68% and 1.64%, respectively.All extracts showed antibacterial activity against pathogenic bacteria [7].Cholestane and dihydropyrene were isolated and identified from C. inophyllum leaves using a crude extract obtained via maceration [25].According to Abbas et al. [48], extracted and fractionated bioactive compounds from fresh fruit peel exhibited antioxidant and antidiabetic activities.Maceration extraction resulted in the highest crude extracts using methanol (101.387g) as a solvent, followed by ethyl acetate (66.929 g) and n-hexane (64.215 g).They also reported that the n-hexane sub-fraction contained a high amount of total phenolic (196.57µg GAE/mg extracts) and flavonoid (38.06 µg QE/mg extracts) content, which showed antioxidant and antidiabetic activity with 6.31% inhibition of DPPH and 16.99% inhibition of α-glucocidase enzyme at 100 µg/mL, respectively [48].
Using the percolation method and optimizing the extraction conditions, Hapsari et al. [24] extracted the highest total phenolic compounds (289.12 mg GAE/g of leaf residue) and total flavonoid compounds (410.4 mg QE/g) using 80% methanol in water at 30 • C for 48 h.Under these conditions, the leaf extracts of C. inophyllum show antioxidant activity, with an IC 50 value of 0.054 mg/mL [24].Navyasri et al. [22] reported maximum phytoconstituents, along with flavonoids in ethanolic leaf extracts obtained using the Soxhlet method, which exhibited moderate-to-highly anti-inflammatory and anti-arthritis activity.Soxhlet extraction is the most conventional and reliable technique for seed oil extraction practice as of today.In another study, oil recovery from C. inophyllum was compared between the screw press and solvent (n-hexane) extraction techniques.Grated kernel with a 14.4% moisture content produced the highest yield (86.4%) using solvent extraction, while oil yield of 78.0% was achieved using the screw press technique [52].
Essential oils from different parts (leaf, stalk, flower, seed, pod, peel, stem wood, stem bark, root wood, and root bark) of C. inophyllum Linn were extracted via hydro-distillation, which yielded (w/v) the highest content in the peel (0.560%) and lowest in the roots (0.219%).Among different plant parts, pod essential oils showed strong α-amylase and α-alpha glucosidase inhibition activity compared to the others parts.The high inhibition efficiency must be triggered by the presence of bioactive constituents against postprandial hyperglycemia and other diseases associated with diabetes mellitus [36].

Non-Conventional Extraction Technique
Very limited research has been conducted for the extraction of bioactive compounds from C. inophyllum using non-conventional methods (Table 2).Nguyen and Tran [50] extracted fatty acids and coumarins compounds from the seeds of C. inophyllum using supercritical carbon dioxide, and investigated their antibacterial and anti-inflammatory activities using the diffusion method performed on white mouse ears (Swiss albino).The fatty acid content in tamanu oil extracted using sCO 2 (72.62%) was higher than that using the Soxhlet (71.18%) and cold press (71.38%) methods.On the other hand, the coumarin (C 9 H 6 O 2 ) content in the tamanu oil extracted using sCO 2 was the highest (169.69µg/g), being 1.79 and 2.04 times as much as that using the Soxhlet method and commercial oil, respectively [50].Fruit peels from C. inophyllum were extracted using the pressurized liquid extraction method, and the phenolic compounds contained in the peels demonstrated promising antibacterial activity against Staphylococcus aureus and Mycobacterium smegmatis [30].Pressurized liquid extraction yielded 9.98% (w/w) crude extract, which was more than double compared to Soxhlet extracts (4.18% w/w) [30].Thy et al. [51] reported higher wound healing properties of tamanu oil extracted using the ultrasound-assisted technique when tested on Swiss albino mice and compared with commercial oil.They achieved an optimal yield of 55.15% when extracting at 42 • C with the time of 23 min and solvent-to-material ratio of 26 to 1 (v/w) mL/g.However, the ultrasound-assisted technique, microwave-assisted extraction, enzymatic extraction, and supercritical fluid extraction were recently used besides Soxhlet for the bio-diesel production from the seeds of C. inophyllum [53].Chemical extraction using hexane as a solvent is very effective compared to mechanical extraction using the screw press.Jahirul et al. [54] investigated the oil extraction efficiency between screw press and solvent (n-hexane) extraction, and reported that the percentage yield is higher in solvent extraction (51%) compared to screw press (~25%).Manto et al. [55] reported that the oil yield via ultrasound-assisted extraction with n-hexane and petroleum ether was 60% better compared to 56% and 30% for Soxhlet and traditional solvent extraction by stirring, respectively.A higher oil recovery from the seeds of C. inophyllum was found using the microwave hydro-diffusion gravity method (3.25% dry wt basis) when compared with the Soxhlet extraction method (2.28% dwb) [56].However, a high content of gum in the extracted oil using these methods causes low-quality physical properties of the bio-diesel production.Rajendran and Gurunathan [39] used ultrasonic waves in conjunction with an equal solvent ratio of diethyl ether:ethanol, producing a maximum-yield oil extraction.The highest percentage of bio-oil, i.e., 82.8% (w/w), was reported under optimized conditions, which included 45 min, a temperature of 36 • C, a seed kernel-to-solvent ratio of 1:15 (w:v), and 54% ultrasonication power.The ultrasound-assisted extraction of oil from C. inophyllum Linn seeds was studied, and the effects of the factors (solvent, extraction time, ultrasonic power, extraction temperature, and liquid-to-solid (L/S) ratio) on the oil yield were optimized by Fuad and Don [57].The highest oil yield (55.44 ± 0.53%.) was obtained using n-hexane as a solvent, extraction time of 20 min, ultrasonic power of 210 W, extraction temperature of 40 • C, and L/S ratio of 20 mL/g [57].

Future Prospects for Effective Extractions
Since C. inophyllum is a rich source of many bioactive compounds (Table 1), it would be difficult to extract all these compounds using an optimized technique or under optimal conditions.Various conventional methods have been employed to extract bioactive compounds from C. innophyllum, as discussed in Section 5.1.Several drawbacks of conventional methods, such as lengthy extraction durations, high temperature requirements, poor extraction efficiency, and the thermal destruction of thermolabile chemicals [44,45], have encouraged growing interest, particularly in green and alternative non-conventional extraction technologies such as SFE.There are several key advantages offered by SFE over conventional methods, including higher purity extracts, faster separation, selective fractionation, single-step processing, no residual solvent, and environmentally friendly procedures [46].To the best of my knowledge, no research has been published on the use of SFE for extracting bioactive compounds from the leaves, stems, roots, and flowers of C. inophyllum.Very limited research has been conducted on the extraction of bioactive compounds from the fruits of C. inophyllum using advanced methods (Table 2).In several of our previous studies, we have reported higher bioactivities of compounds extracted via supercritical fluid compared to conventional methods [58][59][60].Pereira et al. [61] reported that the combination of supercritical and pressurized fluids extraction methods resulted in better extraction performance and identification of more fatty acids, along with a higher TPC, antioxidant activity, and total carotenoids when compared to conventional extracts of Butia capitata fruit pulps.No comprehensive studies have been found for the optimization of extraction parameters (pressure, temperature, flow rate, co-solvent ratio) using SFE and the comparison of their bioactivities among different parts of the plant, influence of geographical location, and harvesting time on the yield quality.Other non-conventional methods, such as microwave-assisted, enzymatic, and ultrasound-assisted methods, could be applied to optimize the extraction technique and conditions to compare the bioactivities of the extracted yield.Mathematical models, such as response surface methodology, could be applied to understand the effect of multiple factors and their interaction on the expected yields.Nevertheless, more research is needed to develop a cost-effective green extraction technology for separating bioactive compounds from the leaves, stems, roots, and fruits of C. inophyllum, evaluate their toxicity and other chemical characteristics, and identify their uses as functional components or medicinal agents.

Conclusions
This review provides information on ethnobotanical and ethnomedicinal use, and conventional and non-conventional techniques for the extraction of major bioactive compounds and pharmacological activities of C. inophyllum.Yield recovery and their bioactivities are primarily influenced by the extraction technique, extraction conditions, and solvent polarity.Maceration is the most common extraction methods used for the phytochemical studies of C. inophyllum.Green technology, such as SFE using response surface methodology, could be used to optimize the extraction conditions for obtaining high-quality yields, rich with bioactive compounds, within a short time.Furthermore, extraction conditions for the combination of advanced techniques, such as SFE with UAE, SFE with PLE, and enzymebased-ultrasound-microwave-assisted extraction, need to be optimized and developed at the pilot scale in order to recover all the potential biologically active compounds from C. inophyllum for commercial uses.
Funding: This study was funded by the USDA NIFA Research Capacity Fund (Hatch) (7005056).

Table 2 .
Extraction technique, conditions, and solvent used for the extraction of bioactive compounds from C. inophyllum.