Pharmacognostical and Phytochemical Studies and Biological Activity of Curculigo latifolia Plant Organs for Natural Skin-Whitening Compound Candidate

Curculigo latifolia (family Amaryllidaceae) is used empirically for medicinal purposes. It is distributed throughout Asian countries, especially Indonesia. This study aimed at standardizing the C. latifolia plant, analyzing its phytochemical profile, and evaluating its pharmacological effects. The powder from each organ (root, stem, and leaves) was standardized organoleptically and microscopically. Samples were extracted by graded maceration using hexane, ethyl acetate, and ethanol. The extracts were determined for total phenolic content (TPC) and total flavonoid content (TFC). Antioxidant (radical scavenging and metal ion reduction) and antityrosinase activities were determined by spectrophotometric methods. Extracts were analysed for phytochemical profiles by LC-ESI-MS. The highest TPC and TFC were found in the ethanolic extract of the root organ (68.63 ± 2.97 mg GAE/g) and the ethyl acetate extract of the stem (14.33 ± 0.71 mg QE/g extract). High antioxidant activities were found in the ethanolic root extract (20.42 ± 0.33 µg/mL) and ethanolic stem extract (45.65 ± 0.77 µg/mL) by DPPH• and NO• assays, respectively. The ion reduction activity (by CUPRAC assay) was most significant in the ethyl acetate stem extract (390.42 ± 14.49 µmol GAEAC/g extract). Ethanolic root extract was the most active in inhibiting tyrosinase (IC50 value of 108.5 µg/mL). The correlation matrix between TPC and antioxidant activities showed a moderate to robust correlation, whereas the TPC and antityrosinase activity showed a robust correlation. The TFC and antioxidant or antityrosinase activities showed a weak to moderate correlation. The LC-ESI-MS data identified major phenols in the active extracts, including methyl 3-hydroxy-4-methoxy-benzoate, quercetin, 4-O-caffeoylquinic acid-1, and curculigoside. Overall, this study suggests that extracts from the C. latifolia plant offer potent antioxidant and antityrosinase activities, allowing them to be used as natural antioxidants and candidates for skin-lightening compounds.


Introduction
Te skin is the outermost layer of the human body.Excessive sun exposure stimulates the formation of melanin pigments.Melanin is the main pigment responsible for human skin, hair, and eye pigmentation.Melanocytes produce melanin through melanogenesis.Melanogenesis and skin pigmentation are the main photoprotective factors in response to harmful UV radiation from sun exposure and skin photocarcinogenesis.Melanin loss and irregular depigmentation loss can represent serious aesthetic and dermatological problems on the face in humans [1][2][3].Conversely, increased melanin synthesis and excessive accumulation of melanin pigment can cause various skin diseases such as acanthosis nigricans, cervical poikiloderma, melasma, periorbital hyperpigmentation, lentigines, neurodegeneration associated with Parkinson's disease, skin aging, and risk of skin cancer [3,4].
During melanogenesis in the skin, tyrosinase is an important enzyme in melanin synthesis.Tyrosinase is also a multifunctional copper-containing metalloenzyme with binuclear copper ions and acts as a reaction accelerator in melanin synthesis.In addition, the obvious efect of melanogenesis due to the hyper-reaction of tyrosinase is the formation of hyperpigmentation of the skin [5,6].Tyrosinase is the main cause of unwanted tanning due to the overproduction of melanin.Terefore, control of enzyme activity by tyrosinase inhibitors is important to treat hyperpigmentation, especially in skin tissues, to keep the skin white and glowing.Many efective inhibitors have been identifed and developed in medical and cosmetic skinlightening products.However, in medicine, only a few compounds are known to be potent and safe tyrosinase inhibitors [3,7].
Te search for bioactive compounds provides relatively safe biological activity through plant development.Since plants are a rich source of bioactive chemicals, most of which are free from harmful side efects, there is an increasing interest in fnding skin-lightening products from natural sources.One of the plants that can be developed as a candidate for skin-lightening compounds is Curculigo latifolia.C. latifolia or Molineria latifolia (Dryand.ex.W.T. Aiton) is a plant of the Amaryllidaceae or Hypoxidaceae family, widely distributed in China, Japan, Nepal, Malaya, India, Australia, and Africa.Te original distribution of this plant came from India, Myanmar, Tailand, the Philippines, the Malaysian Peninsula, Singapore, and especially Indonesia, such as Sumatra, the Lingga Islands, the Bangka Island, Borneo, Java, and Celebes [8][9][10].In diferent regions of Indonesia, C. latifolia plants are also known as marasai, lumbah, marasi, congkok, and doyo.Parts of the C. latifolia plant, such as leaves, roots, fruits, and fowers, have been empirically used by the community as a traditional medicine to treat bloody urine, wounds, constipation, hemorrhoids, and fever and as a source of energy.Additionally, people in Kalimantan, Indonesia, use the leaves and roots of C. latifolia to cure jaundice or hepatitis B [8,10].
Scientifcally, C. latifolia has been reported to have antioxidant [9][10][11][12][13], ultraviolet protection [14], antidiabetic [8,10,11,15], and antibacterial [9] efects.Although scientifc extracts from plants have not been described as antityrosinase, their bioactivity as antioxidants and UV protection establish a pathophysiological relationship with their activity as antityrosinase, making it possible to develop them as candidates for skin-lightening ingredients.Te bioactivity of C. latifolia is strongly supported by the content of secondary metabolites found in these plants.Some information showed that the roots of C. latifolia contain phenolic compounds such as phloridzin, pomiferin, scandenin, and mundulone [12].Te study by Umar et al. [10] also reported that C. latifolia contains phenolic glycosides such as orchioside derivatives, curculigoside derivatives, and triterpenes (cycloartane) groups, namely, curculigosaponin derivatives.Additional information has also been reported that the roots of the C. latifolia plant contain favonoid derivatives such as isorhamnetin, apigenin, hesperetin, and quercetin [16].Te leaves have been reported to contain phenolic glycosides and saponin derivatives (cycloartane triterpenes).Other studies have also revealed that neoculin and curculin compounds have been isolated from C. latifolia fruit and showed diferent pharmacological activities [17,18].
Te presence of phenolic glycosides and favonoid derivatives in C. latifolia has been previously reported so that bioactivity as an antityrosinase can be developed.Several studies have shown that the presence of phenolic and favonoid derivatives in natural products signifcantly inhibits tyrosinase activity.Te number of OH groups in phenolic and phenolic hydroxyl groups in the A and B rings of favonoids can enhance the inhibitory efect on tyrosinase [19].C. latifolia plants are a rich source of phenolic compounds and favonoid derivatives, allowing the development of active skin-lightening ingredients.
Current research aims at investigating the biological activities of the C. latifolia plant organs, including their antioxidant and tyrosinase inhibitory activities.Microscopic characterization and phytochemical screening of plant parts were also undertaken.Te knowledge gained provides important information for the development of C. latifolia as a raw material for medicines and cosmeceuticals.

Plant Material.
Te plant parts used in this study were the roots, stems, and leaves (Figure 1) of Curculigo latifolia.Tey were obtained from the Indonesian Medicinal and Aromatic Crops Research Institute (IMACRI), Menteng, Bogor Baru District, Bogor City, West Java (6 °57′70.2″S;106 °78′62.5″7Ealtitude 15 m).Te specimen was collected in August 2021 and identifed at the Herbarium Bogoriense Biodiversity Research Organization, Biological Research Center Bogor City, West Java, Indonesia.Te process of harvesting C. latifolia plants was carried out at 07-08 in the morning before the plants experience active metabolism due to the infuence of photosynthesis, so the chemical content of the plants is more stable and does not experience decomposition [20,21].

Organoleptic and Microscopic Evaluation.
Organoleptic evaluation was performed on each organ of C. latifolia plants based on sensory parameters including the shape, color, smell, and taste.Microscopic evaluation was performed on 2 Te Scientifc World Journal C. latifolia plant organ powder.Sample powder was placed on a glass object and sprinkled with chloral hydrate as a coloring agent.Te samples were then examined for microscopic parameters using a binocular microscope (XSG, WF10x) at a magnifcation of 400x [22].

Sample Extraction.
Te roots, stems, and leaves of the C. latifolia plant were dried and powdered, followed by gradual maceration using various solvents with diferent polarity levels [23].In the present study, each plant organ (250 g) was soaked with n-hexane at a ratio of 1 : 10 (powder (g): solvent (mL).Te maceration was carried out for 1 × 24 hours at room temperature; thereafter, the macerated sample was fltered.Te residue was remacerated with the same solvent until a clear fltrate was obtained.Te residue of each plant part was then macerated with ethyl acetate solvent in the same way as with n-hexane solvent.Te extraction process was carried out similarly using 70% ethanol.Each pooled fltrate obtained was evaporated using a rotary vacuum evaporator (Buchi, R-100) to obtain a thick extract.

Determination of Total Phenolic Content (TPC) and Total
Flavonoid Content (TFC).Analysis of the TPC and TFC of each organ extract of C. latifolia was carried out by colorimetry methods using a visible spectrophotometer (Shimadzu UV-1900, Kyoto, Japan) [25].In the TPC procedure, an aliquot (0.1% w/v, 0.5 mL) of each extract solution was mixed with 0.4 mL of Folin-Ciocalteu (0.2 M) reagent.Te mixture was allowed to stand for 3 minutes, and then, 2 mL of Na 2 CO 3 7.5% (w/v) was added.Te volume was adjusted to 5 mL by the addition of distilled water.Te mixture was then homogenized and incubated for 30 minutes at room temperature.Te absorption of the sample solution was measured at 635 nm (spectrophotometer UV-Vis, UV-1900, Kyoto, Japan).Te TPC of each extract was calculated using the standard curve equation of gallic acid (2-10 µg/mL).Te TPC of each extract was expressed as gallic acid equivalent (mg GAE/g extract).
Te procedure for determining TFC was as follows.Extract solution (0.1% w/v, 0.5 mL) was mixed with sodium acetate (1 M, 0.1 mL).Te mixture was left for 5 minutes to equilibrate, and then, AlCl 3 (10% w/v, 0.1 mL) was added.Te volume was made up to 5 mL with ethanol and incubated for 5 minutes at room temperature.Te absorption was read at 430 nm using a spectrophotometer.Te TFC of each extract was calculated using the standard curve equation of quercetin (2-10 µg/mL).Te TFC of each extract was equivalent to quercetin (mg QE/g extract).

Determination of Antioxidant Activity.
Diferent in vitro methods were used to determine the antioxidant activity of each extract.Nitric oxide (NO•) and DPPH• radical scavenging assays were carried out to observe the ability of each extract to inhibit the action of radical species, whereas the cupric ion reducing antioxidant capacity (CUPRAC) assay was carried out to observe the ability of each extract to reduce metal ions.

NO Radical Reduction Assay.
Te NO• radical reduction method was performed based on a reported method by Syamsu et al. [26] with slight modifcations.A series of samples (10-1000 µg/mL) were prepared in 0.5 mL of sodium nitroprusside (10 mM in phosphate bufer saline solution).Te mixture was then incubated at 27 °C for 2.5 hours.Te mixture was then added to 1.5 mL Griess reagents which consisted of 750 µL of sulfanilic acid (0.33% b/v in 20% v/v acetic acid glacial) and 750 µL of naphthalene (0.1% b/v).Each mixture was made up to a 5 ml volume with phosphate bufer saline solution and was further incubated for 30 minutes at room temperature.Te absorption was measured at 535 nm using a UV-Vis spectrophotometer.Te percentage inhibition of NO• radicals was calculated using the following formula: Te antioxidant activity was expressed by inhibition concentration 50% (IC 50 ), i.e., the sample concentration that can reduce NO• radicals by 50%.

DPPH Radical Reduction Assay.
Te DPPH• radical reduction method was assayed based on a method described by Nur et al. [27] with slight modifcation.A series of samples (10-1000 µg/mL) was prepared.To each sample was added 1 mL of 0.4 mM DPPH• solution.Te volume was made up to 5 mL in ethanol, and the mixture was vortexed to homogenize.Te sample solution mixture was incubated for 30 minutes in the dark at room temperature.Te absorbance of the sample solution was measured at 516 nm on a UV-Vis spectrophotometer.Te percentage inhibition of DPPH• radicals was calculated using the following formula:  [28,29].A volume of each extract solution was reacted with 1 mL of CuCl 2 reagent (10 mM), 1 mL of neocuproine (7.5 mM), and 1 mL of ammonium acetate (1 M).Te volume was made up to 5 mL with distilled water.Te solution mixture was incubated for 30 minutes, and the absorbance was read at 450 nm by a UV-Vis spectrophotometer.A calibration curve was generated using quercetin standard solution.
Te antioxidant capacity of each sample was expressed as µM gallic acid equivalent antioxidant capacity of extract (µM GAEAC/g extract).Te GAEAC value was calculated using the following formula: Te Scientifc World Journal GAEAC � x value X (1/1000)X sample volume X dilution factor weight sample (g) . (3)

Antityrosinase Evaluation Activity.
Te inhibitory activity against tyrosinase of root, stem, and leaf extracts of C. latifolia was determined colorimetrically based on the modifcation of previous methods [30,31].Te reaction mixture in a 96-well microplate (Iwaki Pyrex) consisted of 80 µL of phosphate bufer (50 mM, pH 6.5), 40 µL of extract solution (1-500 µg/mL), 40 µL of L-DOPA solution (4 mM), and 40 µL of mushroom tyrosinase solution (75 U/mL).Te solution mixture was shaken for 60 seconds and incubated for 30 minutes at 25 °C.Te absorbance was measured using a microplate reader (Promega GloMax) at 490 nm.Te blank solution in phosphate bufer was prepared in the same way as the sample solution.Control samples and blanks were made without the addition of the tyrosinase enzyme.Te following equation was used to calculate the percentage of tyrosinase inhibition: where A is the absorbance of the blank solution with enzymes (blank), B is the absorbance of the blank solution without enzymes (blank control), C is the absorbance of the sample solution with enzymes (sample), and D is the absorbance of the sample solution without enzymes (control sample).Te IC 50 value was calculated using a linear regression equation generated from plots of sample concentration versus percentage of inhibition (% inhibition).

Te Phenolic Composition of the Active Extract by LC-ESI-MS Study.
Analysis of the compounds of extracts was carried out using a liquid chromatography-mass spectroscopy (LC-MS) system using the modifed methods in [32,33].A Waters Acquity UPLC I-Class equipped with a Xevo G2-XS QToF mass spectrometer with an ESI source (capillary 2 kV, temperature 120 °C) was used to generate mass spectra.Sample separation was conducted using an Solvent mixtures used consisted of eluent A (H 2 O + 0.1% formic acid) and eluent B (acetonitrile + 0.1% formic acid), with a ratio of 95 : 5 and 0 : 100.Samples were prepared by dissolving 5 mg of solid, followed by fltering through a 0.22 µm nylon flter.Te Sample solution (5 µL) was injected.Mass fragmentation of phenolics was identifed using the spectrum database (UNIFI) of organic compounds in the instrument application.

Statistical Analysis.
All data were obtained from triplication (n � 3) and expressed as mean ± SD.Signifcant diferences between mean values were analysed by one-way analysis of variance (ANOVA, p < 0.05) using SPSS 23 version.Te TPC and TFC data correlations with antioxidant and antityrosinase activities were analysed using Spearman's correlation by Minitab 20 version.

Organoleptic and Microscopic Analysis. Each organ of
C. latifolia generally has diferent organoleptic properties.In particular, each organ has a distinctive smell.In addition, the root organ tasted bitter compared to other organs.Te diference may be due to the levels of phytochemicals in the diferent organs, leading to diferent organoleptic results, especially in smell and taste (Table 1).Microscopic analysis was performed to provide the standardization parameters of C. latifolia plant organs (Figures 2-4).Microscopic analysis was performed using Figure 3 shows a microscopic form of the stem organ of the C. latifolia plant.Te fragments identifed were trichomes (Figure 3(A)) containing glands with unicellular head stalks.Te stem organ powder of C. latifolia contained xylem vessel fragments thickened into a helical and annular vessel containing fbers (Figures 3(B)-(D)).Te fragment of the periderm (Figures 3(E and F)) was part of the diferent covering tissues than the epidermis in the stem organ.Tis fragment serves to protect/cover the underlying tissue.Te periderm replaces its position when the epidermis is pushed outward, damaged, and peeled of.Te fragments identifed showed that the stem organs in C. latifolia plants underwent secondary growth.Other fragments found in C. latifolia stem organs were endosperm containing oil droplets and aleurone grains containing calcium oxalate crystals (Figure 3   Te Scientifc World Journal oxalate crystals were also seen.Te form of the potassium oxalate found was not determined microscopically.Another characteristic feature identifed is the presence of unicellular trichomes and the absence of glands (Figure 4(d)).

Phytochemical Profle.
Te profle of phytochemical compounds from each C. latifolia plant organ extract was identifed based on colorimetry with specifc reagents.
Table 2 shows the group of compounds from each extract after spraying using staining reagents.Te results in Table 2 show that all extracts of each organ of the C. latifolia plant generally contain a group of phenolic compounds.Similar results were also found in identifying groups of favonoid compounds.Flavonoid compounds have been found in ethanol, ethyl acetate, and hexane extracts from stem and leaf organs.Meanwhile, in the root organ, the favonoid group was present only in the ethanol 6 Te Scientifc World Journal and ethyl acetate extracts, but no results were found in the hexane extract.Generally, positive results were found in identifying steroid/terpenoid groups in all organ extracts except leaf hexane and root ethanolic extracts.Tis group of saponin compounds is found only in the ethanolic extract of roots and stems.Te presence of a group of alkaloid compounds with Mayer's and Dragendorf's reagents was found faintly in each extract, while with Wagner's reagent, it was found only in hexane and ethanol extracts of leaves and stems.

TPC and TFC Analysis.
Te TPC of the root, stem, and leaf extracts of C. latifolia plant organs was determined based on the gallic acid standard curve equation.Te gallic acid concentration was varied to obtain a standard curve equation with a linear regression value of (R 2 ) 0.9942 (y � 0.093 x + 0.0138).Te results can be seen in Table 3.
Te ethanol extracts gave the TPC in the order of root (68.63 ± 2.97 mg GAE/g), followed by the stem (65.95 ± 3.21 mg GAE/g) and leaves (13.59 ± 0.23 mg GAE/ g).A similar trend was observed in the ethyl acetate and hexane extracts, where the root phenol levels were the highest at 64.1 ± 1.19 and 6.35 ± 0.18 mg GAE/g, respectively.Statistically, the TPC of the ethanol extract of the roots was not signifcantly diferent from the ethyl acetate extract of the stems and roots (p > 0.05, n = 3).Tese results correlate well with the phytochemical profle data in Table 2, which shows the phenolic content of the ethanolic extract of roots and stems with a blue intensity.
tTable 3 shows the TFC of each plant organ of C. latifolia.Te ethyl acetate extract of the stems yielded the highest TFC content with 14.33 ± 0.71 mg QE/g, followed by the roots and leaves, 10.35 ± 0.08 and 3.92 ± 0.45 mg QE/g, respectively.Among the ethanol extracts, the leaves had the highest TFC values of 10.21 ± 0.23 mg QE/g, followed by the stems and the roots with TFC values of 7.71 ± 0.15 and 7.44 ± 0.15 mg QE/g, respectively.Meanwhile, the hexane extract of the leaves revealed the highest content of TFC (9.81 ± 0.14 mg QE/g) compared to other organs.Statistically, the ethyl acetate extract of the stems had the highest TFC values and was signifcantly diferent among all extracts (P < 0.05, n � 3, post hoc test LSD).

Antioxidant Evaluation of C. latifolia Extract.
Evaluation of the antioxidant activity of C. latifolia plant organ extracts aimed at investigating the ability of antioxidant compounds in each extract to scavenge free radicals (DPPH and NO scavenging assays) and reduce metal ions (CUPRAC assay).Te antioxidant activities were described as IC 50 values and µmol GAEAC/g extract (Table 4).Based on categorization by Blois [34], the antioxidant power in reducing free radicals was categorized as extreme activity (IC 50 < 50 µg/mL), strong activity (50-100 µg/mL), moderate activity (>100-150 µg/mL), and weak activity (>150 µg/mL).
Te NO• scavenging activities of the leaf extracts (hexane, ethyl acetate, and ethanol) were stronger than those of the stems and roots.IC 50 values of 88.38 ± 0.29 µg/mL (categorized as strong), 25.51 ± 0.58 µg/mL (extreme category), and 22.57 ± 0.74 µg/mL (extreme category) were obtained for hexane, ethyl acetate, and ethanol extracts, respectively.Te root extracts showed a diferent trend for each extract, with the ethanol extract (84.60 ± 0.56 µg/mL) having a strong potential and ethyl acetate (123.47 ± 0.52 µg/ mL) having a moderate potential for reducing NO• radicals.In contrast, the hexane extract of the root organ shows a weak potential with an IC 50 value of 372.65 ± 0.58 µg/mL.Te potential antioxidant activities revealed signifcantly diferent efects (p < 0.05, post hoc test, LSD) for each extract from diferent plant organs.Te total NO test showed that the ethanol and ethyl acetate extracts of the leaf organs and the ethanol extract of the stems showed extreme activity compared to other extracts.Te ethyl acetate and ethanol extracts from leaf organs had the same category of quercetin antioxidant activity as a positive control (3.59 ± 0.072 µg/ mL), which was very potent.Previous studies have not reported the antioxidant potential of each extract and organ of the C. latifolia plant in reducing NO• radicals.Terefore, the results obtained cannot be compared.
Te DPPH• scavenging activities of the plant organs showed a slightly diferent trend than the NO• scavenging activity.Extreme scavenging activities were observed for the ethanolic root and stem extracts and the root ethyl acetate extract with IC 50 values of 20.42 ± 0.33, 41.19 ± 0.32, and 28.30 ± 0.55 µg/mL, respectively, with the activity category (extreme category) of the extracts being the same as that of Unlike the NO• and DPPH• scavenging methods, the antioxidant potential in the CUPRAC method is based on the ability of antioxidant compounds in the sample to reduce Cu 2+ ions to Cu + ions.Te magnitude of the reducing power corresponds to that of gallic acid.Te greater the reducing power, the stronger the antioxidant potential in a sample.Te reducing power of each extract is shown in Table 4.All ethyl acetate extracts from each organ showed higher reducing power, followed by ethanol extracts from each organ of the C. latifolia plant.All plant organs found the lowest reducing power in the hexane extract samples.Signifcant intersample antioxidant activity was detected in the ethyl acetate extract (390.42 ± 14.49 µM/g extract) of C. latifolia leaves (p < 0.05, post hoc test, LSD, n � 3).Te antioxidant potential of ethyl acetate leaf extract in the CUPRAC method correlated with the antioxidant potential of the ethyl acetate leaf extract with an extreme category of the NO• radical scavenger method.To date, no study has reported yet on the antioxidant activity of extracts from the leaves, stems, and roots of the C. latifolia plant, including the radical scavenging and metal ion reduction activities.

Antityrosinase Evaluation of C. latifolia Extract.
Root ethanol extract had the lowest IC 50 value of 108.5 µg/mL, indicating strong tyrosinase inhibitory activity compared to other samples.However, the hexane extracts of leaves, stems, and roots showed no tyrosinase inhibitory activity with IC 50 values at concentrations >1000 µg/mL (IC 50 values obtained from exploration data).Te ethanol extracts of the stems and leaves and the ethyl acetate root extract showed moderate inhibitory activity.IC 50 values <100 µg/mL indicated a strong inhibitory potential, IC 50 values of 100-450 µg/mL a moderate inhibitory potential, and IC 50 values of 450-700 µg/mL a weak inhibitory potential.Kojic acid as a positive control achieved an IC 50 value of 5.55 µg/mL with a strong potential compared to C. latifolia root ethanolic extract.However, the ethanolic extract of C. latifolia root still has a strong potential to inhibit tyrosinase activity, so it can be considered a candidate for skin lightening (depigmentation).

Te Phenolic Composition of the Active Extract by LC-ESI-MS Study.
Extracts from C. latifolia, which were found to have moderate to strong antioxidant and antityrosinase activities in vitro, were assayed for phenolic compound content.Terefore, the ethyl acetate root extract and the ethanol extract of the roots, stems, and leaves were analysed for their phenolic compounds using the LC-ESI-MS method.Table 5 shows the group of phenolic compounds identifed based on LC-ESI-MS analysis.
In each extract, the presence of phenolic compounds in the form of methyl 3-hydroxy-4-methoxybenzoate (183.06 m/z), quercetin (303.05m/z), 4-O-cafeoylquinic acid-1 (355, 10 m/z), and curculigoside (489.14 m/z) was identifed in the ethyl acetate root extract and the ethanol extract from leaves, stems, and roots, respectively (Figure 5).Quercetin and curculigoside were also identifed in the ethanolic extract of C. latifolia leaves and roots by the study of Zolghadri et al. [3] using the UHPLC-Q-Orbitrap-HRMS approach.However, the compounds methyl 3-hydroxy-4methoxybenzoate and 4-O-cafeoylquinic acid-1 identifed in the ethyl acetate root extract and the ethanol stem extract provide additional information regarding the class of phenolic compounds present in C. latifolia.

Discussion
Tis study tested the antioxidant and antityrosinase activities of C. latifolia extracts.Tis study also provides supporting information on the microscopic properties and screening of phytocomponents from each plant organ extract to supplement the scientifc data on these plants.Although previous studies have reported on the phytochemical screening and antioxidant evaluation, the available information is limited to one part of the plant.Hence, the current research has provided more thorough information on various plant organs, including roots, stems, and leaves.In addition, this study also examined the phytochemical and bioactivity profles of several extracts using solvents such as n-hexane, ethyl acetate, and ethanol-aqueous (70% v/v).Previous studies examining C. latifolia have primarily focused on its bioactivity as an antidiabetic and antibacterial agent.Te present work is the frst to report on the antioxidant and antityrosinase activities and the association between these bioactivities.
Te reported organoleptic and microscopic properties of C. latifolia are part of the standardization parameters for raw materials.Organoleptic and microscopic assessments identify the purity, safety, and quality assurance of natural raw materials to ensure that these raw materials are not added to synthetic raw materials when used as standardized herbal medicinal products [22,35].Te organoleptic evaluation of each plant organ was based on sensory analysis to identify shape, color, odour, and taste (Table 1).Microscopic analysis of the dry powder of each plant organ revealed diferences in each fragment.Some of the identifed fragments have their uniqueness.Terefore, the results of the microscopic analysis can provide an overview of the characteristics of C. latifolia plants that have not yet been reported.Te results of an organoleptic and microscopic analysis can help to assess the purity of natural medicinal products [36].In phytochemical screening studies, C. latifolia extract has been shown to generally contain phenolic, favonoid, and steroid/terpenoid compounds (Table 2).Several studies have shown that the roots and leaves of C. latifolia contain phenolic glycoside compounds such as curculigoside, orcinol glycoside, phloridzin, pomiferin, scandenin, and mundulon [3,5].In addition, the roots and leaves also showed the presence of steroid/terpenoid compounds such as curculigosaponin and cycloartane triterpene [10].Te present study confrmed the previous reports.It is worth noting that no chemical analysis of the stem organs of C. latifolia has been reported.Te presence of C. latifolia chemical compounds can provide a further understanding of their pharmacological bioactivities.
Plant organs may contain diferent phenolic compounds [37][38][39].However, the extraction solvent afects the ability to extract diferent phenols from each plant organ.In general, this study showed that the root organs of C. latifolia had higher phenolic content than other organs (Table 3).Tis study also adds to the data that ethanol solvents can extract phenolic compounds.Compared to other solvents, ethanol can extract the most extensive phenolic compounds.Te high polarity of phenols results in a more efective extraction of phenols by polar solvents such as ethanol than other solvents [40].
Similarly, studies by Umar et al. [10] showed that the ethanolic extract of the roots provided high levels of phenols compared to the ethanolic extract of the leaves.Determination of TPC in the stem organs of C. latifolia plants has not been reported.Terefore, this study complemented the data related to TPC in other organs of C. latifolia plants.In the TFC test, C. latifolia extract was reported to have diferent concentrations in each organ.Te diferent profles of favonoid content indicate that each part of the plant has a diferent accumulation of favonoid content.Studies by Umar et al. [10] also reported similar results that the leaf organ ethanol extract had a higher TFC than the root ethanol extract.However, no data on the TFC of the ethyl acetate and hexane extract of C. latifolia have been reported.Te results of the present study can provide an overview of comparisons of TFC levels using diferent solvents and plant organs of C. latifolia.
A plant's high levels of total phenols and favonoids also ofer great potential for its antioxidant activity.C. latifolia plants are supported by high levels of phenols and favonoids in every part of the plant.Flavonoids, the main phenolic group, play a role in biological activity, including their great activity in reducing various free radicals.Te more hydroxy groups attached to the phenolic aromatic nucleus provide greater reducing power towards free radicals.Several previous studies have shown that the antioxidant activity is increased due to the presence of phenolic hydroxyl in the A and B rings of the favonoid skeleton [41,42].Tere are several reasons that the more phenolic hydroxyl groups present in a compound, the greater the antioxidant efect.For several reasons, the more hydroxyl groups, the more hydrogen is donated to free radicals to stabilize the radical reaction.Te phenolic hydroxyl group exerts a strong electronic efect on the radicals, making it easier to neutralize the radical reactions [19,43].
Te presence of phenolic and favonoid compounds from C. latifolia plants supports the antioxidant activity.Te antioxidant activity in this study showed that the ethanol and ethyl acetate extracts from each organ of the Te Scientifc World Journal C. latifolia plant tended to provide strong to very strong antioxidant activity as tested by three diferent test methods, including DPPH, NO, and CUPRAC assays.
Testing the bioactivity of C. latifolia extract as an antioxidant tended to give the same results across the three methods used in this study.One or two types of extracts   Te Scientifc World Journal that do not correlate with the test of the three methods used may be afected by the chemical content of each extract in terms of content and compound group.
Several studies relevant to the potential of C. latifolia to reduce DPPH radicals showed that ethanol and aqueous extracts from plant roots were the most potent antioxidants [9,10,12,33].In the ethanol extract of leaf organs, the present study showed a strong activity in reducing DPPH.In contrast, results found in the study of [10,33] showed weak potential for reducing DPPH radicals by the ethanol extract of leaf organs.However, no information has been reported on the bioactivity of stem organs and extracts of ethyl acetate and hexane from C. latifolia plant organs.Testing of the antioxidant bioactivity of C. latifolia plants using the NO and CUPRAC methods is still limited, so the informational data obtained can be used as a reference for future research developments.Antioxidant activity testing using DPPH and NO radical scavenging methods and Cu reduction aimed at observing the antioxidant bioactivity profle of compounds from C. latifolia plants.DPPH and NO are free radicals that can be converted by biochemical reactions in the body to modulate the formation of reactive oxygen-nitrogen species (RONS).Te formation of RONS can be accelerated by a catalytic reaction in the presence of metals such as copper, leading to a transfer of copper ions that generates oxidative stress [44,45].Tere was a biochemical mechanism for the formation of RONS,, so the three methods were carried out to fnd out the description of the bioactivity of C. latifolia compounds as antioxidants.
Several studies suggest that excess reactive species, including reactive oxygen species and reactive nitrogen species, aggravate pigmentation, which in turn causes uneven skin tone, pigmentation disorders, roughness, and even aging.In the present study, tyrosinase inhibitory activity was determined to assess the potential of C. latifolia plant organs to inhibit melanin formation when used as a whitening agent in the cosmetics industry [11,35].Mushroom tyrosinase catalyzes the formation of L-tyrosine from L-DOPA, which subsequently forms the dopachrome.When adding an inhibitor in the form of C. latifolia extract, the color of the solution faded due to the inhibitory activity of the tyrosinase enzyme [30,46].Table 6 shows the antityrosinase activities of each C. latifolia organ extract.In vitro, the ethyl acetate extract of the roots and the ethanol extract of the stems and leaves had moderate tyrosinase inhibitory activity.In contrast, the ethanol extract of the roots of C. latifolia showed potent tyrosinase inhibitory activity.
Nonetheless, the kojic acid positive control activity was signifcant, giving it the strongest activity among the extracts.However, it can still be assumed that C. latifolia extract contains compounds that inhibit tyrosinase activity.Te tyrosinase inhibitory activities of root organ ethanol extract were supported by the presence of phenolic and favonoid compounds (Tables 2 and 5), which are responsible for the reduction of tyrosinase activity.Phenol and favonoid derivatives (Figure 5   Te Scientifc World Journal inhibitors [3].Hydroxyl groups in rings A and B of the favonoid compounds inhibitory activity by directly binding to tyrosinase and Cu 2+ chelates as metalloenzymes in tyrosinase.In addition, it is predicted that the formation of the Cu 2+ complex of tyrosinase with the catechol structure (3,4-) of the dihydroxy group on the C-ring of quercetin also afects the inhibition of tyrosinase [46][47][48][49].Te bioactivity of C. latifolia extract provides new information as an antityrosinase that has not been previously reported.
A correlation analysis, adopted from Spearman's method, was performed to ensure a correlation between the research results obtained.Spearman's correlation aims at expressing the strength and direction of the linear correlation between TPC and TFC on antioxidant and antityrosinase activities.Te levels of phenols and favonoids (mg/g extract, n � 3) of each extract were correlated with the IC 50 value (n � 3) of the antioxidant and antityrosinase activities of each extract (Figure 6).Te correlation coefcient plot matrix showed diferent correlations.Te strongest category correlation between TPC and antioxidant activity was found in the DPPH method with r � − 0.917, p < 0.01.Tese results show that the antioxidant activity of each extract is infuenced by 91.7% by the presence of phenolic compounds, while other compounds infuence only 8.3%.Te correlation coefcient of TPC with an antioxidant activity using the CUPRAC and NO scavenger methods was categorized as strong (r � 0.667) and moderate (r � 0.400), respectively.
In contrast, the matrix plot between TFC and antioxidant activity showed weak to moderate correlations.In the DPPH radical reduction method, the correlation of its activity was obtained due to the infuence of favonoid compounds with a medium category (r � 0.494).At the same time, the antioxidant activity of the NO and CUPRAC methods revealed a weak correlation with favonoid compounds (Figure 6).
Te antityrosinase activity of each extract also correlated with the content of phenolic compounds in each sample with a correlation coefcient of r � 0.865 (p < 0.01), while the other compounds afected the rest.However, the presence of favonoid compounds in each extract resulted in a weak correlation coefcient (r � 0.392, p > 0.01) for its antityrosinase activity (Figure 6).However, this study shows that the major compounds in phenols were predicted to contribute to their bioactivity as antioxidants and antityrosinases.

Conclusions
Tis study reported the antioxidant and tyrosinase inhibitory activities of several extracts of the C. latifolia plant.Microscopic studies and the correlation between TPC and TFC with antioxidant and antityrosinase bioactivities were reported for the frst time.It was concluded that the ethanol extracts from the roots, stems, and leaves of C. latifolia provide potent antioxidant and antityrosinase activities and are supported by high levels of total phenolic compounds and favonoids.Te LC-ESI-MS identifed phenolic compounds from C. latifolia plants.Information obtained from the present study could not yet describe the actual mechanisms for the activity of antityrosinase.Terefore, an indepth study of this mechanism is still needed, both in silico and in vivo.We hope that the results of our study provide scientifc information for the development of C. latifolia plants as candidate skin-lightening ingredients and, in the future, can fnd markers that can be used as candidates for efective skin-lightening agents and formulated into cosmetic dosage forms.
(G)).Tis property is also found in many plants.Microscopic analysis of C. latifolia plant root organ powder identifed four fragments of xylem vessels, parenchymal cell fragments, xylem fbers, and trichomes (Figure 4).Te network and helical thickening of the xylem vascular fragments were visualized (Figure 4(a)).Tis section also illustrates the presence of xylem fbers (Figure 4(c)) shaped like long cells with pointed ends.Tracheids were found in the longest cells, but at microscopic magnifcation, the shape of the tracheids cannot be seen in the xylem fber fragments.Parenchymal cells (Figure 4(b)) with potassium

Figure 5 :
Figure 5: Phenolic compound chromatograms and mass spectrum of the ethyl acetate root extract (a), ethanol leaf extract (b), ethanol stem extract (c), and ethanol root extract (d).

Figure 6 :
Figure 6: Spearman's correlation coefcient analysis between TPC and TFC with antioxidant and antityrosinase activities of nine extracts in triplicate.p value <0.01 indicates variable signifcance.TPC: total phenolic content and TFC: total favonoid content.

Table 1 :
Organoleptic evaluation of organ plants from C. latifolia.

Table 2 :
Phytochemical profle of each extract by specifc reagent.
Te Scientifc World Journal the positive control (4.76 ± 0.09 µg/mL).Te ethanol extract of the leaves and the ethyl acetate extracts of the stems and leaves showed potent activity with IC 50 values of 50-100 µg/ mL.In contrast, hexane extracts from leaves, stems, and roots showed a weak efect in reducing DPPH• radicals (IC 50 > 200 µg/mL).

Table 4 :
Antioxidant activity of hexane, ethyl acetate, and ethanol extracts of the leaves, stems, and roots of C. latifolia plants.

Table 6 :
Evaluation of antityrosinase activity of ethyl acetate, and ethanol extracts of the leaves, stems, and roots of C. latifolia plants.