Under-utilized wild fruit Lepisanthes rubiginosa (Roxb.) Leenh: A discovery of novel lycopene and anthocyanin source and bioactive compound profile changes associated with drying conditions

Abstract Lepisanthes rubiginosa (Roxb.) Leenh (LRL), a wild fruit of Thailand, was characterized for phytochemicals including phenolic acids, flavonoids, carotenoids, anthocyanins, organic acids, and sugars. To process it, three drying methods were investigated, namely, freeze drying (FD), hot-air drying (HD), and sun drying (SD), which affected chemical components and antioxidant activities. The predominant phenolic acids and flavonoids were p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, quercetin, rutin, and myricetin. Remarkably high amounts of lycopene (158 mg/100g db) and cyanidin-3-O-glucoside (2005 mg/100g db) were observed. Flavonoids, carotenoids, and anthocyanin were decreased by all drying methods. Overall, FD was considered to be the most suitable for drying LRL fruit. Fructose conversion to mannitol during drying was explored by FTIR spectroscopy analysis in FD and HD samples. This study has revealed new information about LRL fruits, which could be a potential source of bioactive compounds; an appropriate drying method is suggested for further applications.


Introduction
Lepisanthes rubiginosa (Roxb.)Leenh (LRL), in Thai called "Mahuad", is widely grown in Thailand and other Southeast Asian countries, including Indonesia, India, and Malaysia. [1]This plant has been traditionally used in folk medicine to treat various symptoms, such as antipruritic, headache, fever, antitussive, and it is also used as a tonic. [1,2]Previous studies reported that several parts of LRL plants, including leaves, bark, flowers, and fruits, have been associated with many beneficial effects.For instance, extracts of LRL leaves have demonstrated potential antioxidant, analgesic, antihyperglycemic, neuropharmacological, and antidiarrheal activities. [2]The bark extracts may also express antibacterial activity against Staphylococcus aureus, Salmonella Typhi, and Shigella dysenteriae. [3]he essential oil, extracted from the flowers, showed anticancer and antioxidant activities. [4]Essential oils from flowers and fruits also express antimicrobial activities against certain pathogenic microorganisms, including Trichophyton mentagophyte, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. [4]Aqueous extracts of the fruit have been reported to decrease locomotion and improve the thiopental-induced sleeping period. [2]revious research has reported significant bioactive compounds found in different parts of LRL.For instance, the leaf extract was found to contain alkaloids, flavonoids, phenolics, tannins, saponins, etc. [2] The extracts of the bark were reported to contain alkaloids, saponins, phenolic compounds, and carbohydrates. [3]Nerolidol, palmitic acid, and farnesol were the major compounds found in the flower essential oil of LRL. [4]The essential oil extracted from the fruit of LRL showed major compounds including palmitic acid, myristic acid, and linoleic acid. [4]However, bioactive compounds in LRL fruits have not been individually classified, particularly for Thai varieties.
Although the fruit of LRL has long been consumed by local Thai people, it is still underutilized.Usually, the ripe fresh fruit is consumed without undergoing any processing.As it is a seasonal fruit, a process that can preserve the fruit for off-season consumption is needed.One of the common methods to prolong the shelf life of perishable foods is through a drying process, which involves removing moisture from the food products until they reach a certain limit. [5]Various drying techniques have both advantages and disadvantages.Sun drying (SD) is the traditional method with the advantages of being environmentally friendly, cheap, and non-pollutant. [5]However, unpredictable weather and long drying times of SD might affect bioactive compounds.The low and inconsistent temperature of SD might also cause the degradation of bioactive compounds. [6]Hot air drying (HD) is the most common method in drying processes.However, due to the high temperature and long process of drying, the product may undergo changes in chemical compounds and nutritional value, which can affect color degradation, flavor, and bioactive compounds. [5,6]Freeze drying (FD) involves dehydration through sublimation.Due to low temperatures, this method preserves heat-sensitive compounds.However, this technique is expensive and time-consuming.Different conditions of drying methods might affect the bioactive compounds in LRL.Therefore, this research aimed to investigate the phytochemical composition and the biological activity of LRL.In addition, the effects of different drying methods (SD, HD, FD) on bioactive compounds including phenolic acids, flavonoids, carotenoids, anthocyanins, organic acids, and sugars were studied.We are the first to report on the classification of bioactive compounds in LRL.We expected to obtain a suitable drying method for preserving bioactive compounds in LRL as well as useful information for practical use in domestic and commercial applications.

Fruit sources and sample preparation
Samples of the fresh fruit (Lepisanthes rubiginosa (Roxb.)Leenh) were harvested in April-May, 2021, from the northeastern region of Thailand.Plants of this fruit were identified by a plant taxonomist from Walai Rukhavej Botanical Research Institute (Mahasarakham University, Mahasarakham, Thailand).The specimens were also deposited in the herbarium and the voucher specimen number was CT020421.Raw fruits were washed before removing the seed from the pulp.Each 500 g of pulp was dried using either freeze drying (FD), hot air drying (HD 60 C) or sundrying (SD) methods.The drying procedure was performed as described previously [6] except for the HD temperature that the samples were dried at 60 C. The moisture content for each method was less than 7%.For FD, samples were first frozen at À50 C for 12 h, then dried using a freeze dryer (Scanvac CoolSafe model 100-9 Pro, LaboGene ApS, Denmark).The heating plate and cold trap were cooled to À100 C and the absolute pressure of the vacuum was under 20 Pa.For HD, the samples were dried at 60 C [7] using an electric thermo-static drying hot-air oven (model FED 115, WTB Binder, Germany).For SD, the fruit pulp was subjected to sun-drying at 35-45 C for 3-5 days.The dried samples were ground, sieved through a 40mesh sieve wire and stored at À20 C until analyzed.The color of samples was measured in terms of CIE L Ã a Ã b Ã using a CR-400 colorimeter (Chroma Meter; Konica Minolta Sensing Inc. Osaka, Japan).The fruit pulp "without any drying process" (fresh) was kept at 4 C for comparison with the dried samples.

Moisture content
The moisture content was performed according to the AOAC (1995) procedure. [8]Briefly, 3 grams of samples were dried at 103 C ± 1 C to a constant weight using a vacuum oven (UFE 600, Memmert, Memmert Company, Germany).The results were reported as dry mass.

Sample extraction of total phenolics, total flavonoids, total anthocyanins, and antioxidant activities
Bioactive compounds were extracted according to a published method. [6]Briefly, 5 grams of ground samples were extracted with 20 mL of a 80:20 methanol: water mixture.The samples were shaken at 150 rpm for 12 h at ambient temperature.The solvent was collected and the samples residues were extracted again with 10 mL of the same solvent.The first and second extraction were combined and the volume was adjusted to 20 mL with a 80:20 methanol: water mixture.The extracted samples were used to analyze total phenolics, total flavonoids, total anthocyanins, and antioxidant activities.

Total phenolics content (TPC)
Total phenolics content was determined according to a previously published method [9] with some modifications.Briefly, 20 mL samples were mixed with 100 mL Folin-Ciocalteu reagent and incubated at 37 C for four min.The samples were mixed with 75 mL of a 10% sodium carbonate solution.After incubation at room temperature for 2 h, the absorbance of the samples was measured at 725 nm using a microplate reader (Varioskan Lux, Thermo Fisher Scientific, USA).Total phenolics content was expressed as mg gallic acid equivalents/g dry basis (mg GAE/g db).

Total flavonoids content (TFC)
Total flavonoids content was performed using the colorimetric method as described previously [6] with some modifications.In a 96-well plate, 25 mL of the extracted samples, 100 ml of purified water, and 10 ml of a 5% NaNO 2 solution were combined.After shanking for 5 min, 15 mL of a 10% AlCl 3 6H 2 O solution was added to the mixture.The samples were shaken for 6 min, then 50 mL of 1 M NaOH and 50 mL of filtered water were added to the samples.The absorbance of the samples was measured at 510 nm using a microplate reader (Varioskan Lux, Thermo Fisher Scientific, USA).The rutin equivalents per gram of dry basis (mg RE/g db) were used to express total flavonoids content.

Total anthocyanins content (TAC)
Total anthocyanins content was measured using the pH-differential method according to a published method. [10]The results were expressed as mg of cyanidin 3-glucoside equivalent/100 g of dried basis (mg C3G E/100 g db).

Total carotenoids content (TCC)
Carotenoids were determined according to a published method [11] with some modifications.Briefly, 2 g of samples were mixed with 25 mL acetone.After 10 min of agitation, the samples were filtered through Whatman No. 1 filter paper.The filtrate was transferred into a separatory funnel and 20 mL petroleum ether was added.An additional 100 mL of distilled water was used to remove the acetone and the bottom layer was discarded.The process was repeated twice.The layer of petroleum ether was filtered through Whatman No. 1 filter paper covered with 5 g of anhydrous sodium sulfate.The pooled petroleum ether extracts were adjusted with petroleum ether to a volume of 25 mL.The absorbance of the extracted samples was measured at 450 nm using a UV-vis spectrophotometer (ShimadzuUV-1700, Japan).Total carotenoids content was expressed as mg b-carotene equivalent/g of dried basis (mg b-carotene E/g db)

Determination of phenolic acids and flavonoids
The individual phenolic acids and flavonoid compounds were extracted as explained previously. [6]The extracted samples were filtered through a 0.45-mm nylon membrane filter and analyzed by high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) with C18 column (InertsilV R ODS-3; 250 mm Â 4.6 mm i.d., 5 mm, GL Sciences Inc., Tokyo, Japan) followed the published protocol. [9]

Determination of anthocyanin compounds
The anthocyanins were extracted as described previously. [12]The extracted samples were filtered through a 0.22 lm membrane filter and stored at À18 C prior to analysis by LC-MS/MS (Shimadzu LCMS-8030 triple quadrupole mass spectrometer) with ESI mode and HPLC system (Shimadzu, Kyoto, Japan).Eluents consisted of acetic acid 1% (v/v) in deionized water (Solvent A) and acetonitrile (Solvent B).Anthocyanins were eluted using an isocratic condition.The MS/MS condition was performed as described previously [13] for the analysis of anthocyanin.Auto-optimizations are shown in Supplementary data (Table S1).The concentrations of anthocyanins were quantified using external standards.

Determination of carotenoid compounds
The carotenoid contents in samples were extracted using a previously published procedure. [14]The extracted sample solution was filtered through 0.45 lm membrane filters and 20 lL samples were analyzed by HPLC (Shimadzu LC-20AC, Kyoto, Japan).

Determination of sugar and organic acid contents
Individual sugars were analyzed by HPLC (A Shimadzu 20 series system) according to a published protocol. [15]Sucrose was analyzed using an Aminex HPX 87 C column (300 mm Â 7.8 mm; particle size 9 lm, Bio-Rad, Marnes-la-Coquette, France) with a refractive index detector.The monosaccharides (glucose, fructose, sorbitol, and mannitol) and organic acids were analyzed according to a published method [15] using an Aminex HPX 87H column (300 mm Â 7.8 mm; particle size 9 lm, Bio-Rad, France) with a refractive index detector.Detection was performed using a diode array detector at 210 nm.

Fourier transform infrared (FTIR) spectroscopy
The Lepisanthes rubiginosa (Roxb.)Leenh samples were studied using an FTIR spectroscopy instrument with a UATR accessory for Frontier equipped with a Diamond/KRS-5 crystal composite (Perkin Elmer, USA) according to a published method. [6]The program performed an automated subtraction of the recorded background spectrum.

2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging
The DPPH free-radical scavenging of the extracts was performed as described previously [6] with some modifications.In general, 20 mL of the extracts or the control was mixed with 180 mL of 60 lM DPPH solution, incubated at 30 min in the dark at room temperature.The absorbance was measured at 517 nm using a microplate reader (Varioskan Lux, Thermo Fisher Scientific, USA).The results were displayed as mg Trolox equivalents per 100 g dry basis (mg TE/100 g db).
Ferric reducing/antioxidant power assay (FRAP) The FRAP procedures were performed according to the published method [6] with a slight adjustment.In the 96-well plate, 5 mL of extracted samples were mixed with 180 mL of FRAP reagent.The mixture was shaken for 1 min, incubated at 37 C for 15 min and the absorbance was measured at 593 nm.The results were expressed as mg FeSO 4 per g dry basis (mg FeSO 4 /g db).

Statistical analysis
The samples were done in three replicates and all data were expressed as the mean ± standard deviation (SD).
To evaluate a significant difference (P < 0.05), oneway analysis of variance (ANOVA) and the least significant difference (LSD) test were applied.Different letters between samples indicate significant differences (P < 0.05).

Results and discussion
Drying methods affected the chemical composition of Lepisanthes rubiginosa (Roxb.)Leenh (LRL) samples in many ways.Different colors observed in each sample are shown in Supplementary data (Table S2).
Overall, the main bioactive compounds found in the fresh samples were phenolics, flavonoids, and carotenoids.The predominant phenolic acids and flavonoids were p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, quercetin, rutin, and myricetin.Remarkably high amounts of lycopene and cyanidin-3-O-glucoside were observed.In addition, a number of organic acids and sugars were characterized.The phytochemicals and biological activity of fresh LRL associated with the effects of drying methods will be discussed as follows: 3.1.Changes due to drying methods on the TPC, TFC, TAC, TCC, vitamin C, and anti-oxidant capacity The bioactive compounds and antioxidant activities from fresh, HD, FD, and SD treated samples are shown in Figure 1.As can be seen clearly, fresh samples showed the highest values in total phenolics content (TPC), total flavonoids content (TFC), total anthocyanins content (TAC), total carotenoids content (TCC), vitamin C, and antioxidant capacity.The highest value of TPC in fresh LRL was 297 mg GAE/g db, followed by FD, HD, and SD (246, 202 and 184 mg GAE/g db, respectively) (Figure 1a).The amount of TFC, as an indicative bioactive compound, showed the highest content in fresh samples (31 mg RE/g db), followed by FD, SD, and HD (21, 15, 12 mg RE/g db, respectively) (Figure 1b).These results indicated that thermal treatment led to a decrease in TFC and TPC values in fresh samples, which aligns with a previous study. [6]nthocyanins are water-soluble pigments that belong to the phenolic group.The TAC of L. rubiginosa was found to be highest in fresh samples (396 mg C3GE/100 g db), followed by FD, SD, and HD (363, 201, 102 mg C3GE/100 g db, respectively) (Figure 1c).The stability of anthocyanins depends on various factors such as the type of pigment, co-pigments, light, temperature, pH, metal ions, enzymes, oxygen, and antioxidants. [16]Therefore, different drying conditions (temperature, time, light, etc.) might affect the stability of anthocyanins, resulting in varying decreases in TAC.Total carotenoids contents were reduced by all drying methods, with FD being the most suitable method to preserve TCC, while SD was the least appropriate method (Figure 1d).The lowest TCC found in the SD sample is twelve times lower than that of the fresh samples.
Ascorbic acid, commonly known as vitamin C, is present in various fruits.The level of the vitamin C content in fresh samples was 380 mg/100 g db, which falls in the medium range compared to other fruits. [17]hile it has been well established that vitamin C is highly sensitive to heat treatment, our study has found that vitamin C contents of LRL after being dried by FD, HD and SD were not significantly altered as a result of heat.Those values decreased approximately 3.8 times compared to the fresh samples (Figure 1e).[20][21] In this study, the high temperature in HD and the combined exposure to light and heat in SD have the potential to reduce vitamin C content to comparable levels.Freeze drying, on the other hand, has been found to significantly decrease the vitamin C content in certain type of food. [22,23]lthough the freezing process alone typically did not affected the vitamin C content, significantly decrease in vitamin C level was observed in green bean and spinach. [24]Another study conducted on four types of berries revealed that freezing resulted in a 14% reduction in vitamin C content, while during lyophilization (freeze drying), the losses in vitamin C were found to be 84% compared to fresh fruits. [22]The freezing and freeze drying processes of LRL fruit may potentially cause a reduction in vitamin C content and may not be a suitable method to preserve vitamin C in LRL fruit.
In general, thermal processing is expected to reduce bioactive compounds.Our findings are consistent with a previous study, which reported that a longer drying period affected some chemical changes, including the destruction of vitamins and nutrition, pigment oxidation, and solute migration of the food components, along with physical appearance. [6]The alteration of LRL phytochemicals after being processed could be attributed to the inhibition of polyphenol oxidase at a low temperature when using freeze drying. [6]This enzyme could be activated at sun drying temperatures (35-40 C) or still retain its activity at a higher temperature (60 C) of hot air drying. [25]he antioxidant activities in FD, HD, and SD samples showed a significantly decreased value compared to fresh samples (Figure 1f, g).The DPPH radicalscavenging activity of fresh samples was 71 mg TE/g db, whereas the activities of different drying samples ranged from 18 to 19 mg TE/g db (about four times lower than fresh samples) with SD being the highest activity and FD being the lowest activities (Figure 1f).The FRAP value in fresh samples was 187 mg FeSO 4 /g db, while the values in FD, HD, and SD ranged from 90 to 98 mg FeSO 4 /g db (approximately half of fresh samples) with FD having the highest value and SD having the lowest value (Figure 1g).It is important to note that both DPPH and FRAP assays are employed to evaluate antioxidant activities, yet they operate based on different mechanisms.[28] The observed discrepancy can be attributed to the distinct mechanisms through which bioactive compounds react with different assays.Several studies have also reported the variation between the DPPH and FRAP assays, [29][30][31] therefore it is advisable to employ multiple assay methods to comprehensively assess antioxidant capacity to account for the differences and limitations associated with each technique.The changes in antioxidant levels among different drying methods may be attributed to the various drying conditions (temperature, time, light, etc.) that correspond to the loss of antioxidant activity.Phenolics and flavonoids are common bioactive molecules from plants with antioxidant properties. [32]The level of TPC and TFC are therefore associated with antioxidant activities.However, certain bioactive compounds present in drying samples might be deactivated due to the drying methods, causing lower values of antioxidant activities.To fully understand these differences, further studies are needed to evaluate how each drying process affects the mechanism between bioactive compounds related to antioxidant activities.
In regard to drying methods, FD seems to be the most suitable method for preserving most bioactive compounds of LRL in this present study.However, when comparing between HD and SD, the choice depends on the target compounds of interest.For instance, HD may offer more advantages for TPC and TCC, while SD is better for TFC and TAC.Further study could explore the potential influence of alternative drying techniques, such as infrared drying, vacuum drying, or microwave drying, [33][34][35] on preservation of bioactive compounds.

Changes in individual phenolic acids and flavonoids with different drying methods
Table 1 displays the individual phenolic acid contents in LRL as a result of various drying techniques.A total of 13 phenolic acids were monitored, and the average content of each compound varied among the four treatments (fresh, FD, HD, and SD).Total phenolic acids decreased in all samples after drying.The highest content of total phenolic acids was found in the fresh samples, followed by FD, SD, and HD, respectively.The dominant phenolic acids in LRL extracts were p-hydroxybenzoic acid and chlorogenic acid.These two phenolic acids differed in their content among the drying methods.The content of phydroxybenzoic acid was mostly present in fresh samples, followed by FD, SD, and HD, respectively, whereas the content of chlorogenic acid was mostly present in FD followed by fresh, SD, and HD, respectively.Chlorogenic acid is a major phenolic acid in coffee. [26]Chlorogenic acid and its related compounds have been associated with many health benefits, including antivirus activities, anti-hepatitis B virus (HBV) in vitro and in vivo, anti-antidiabetic effects, etc. [26] The higher chlorogenic acid content in FD (compared to fresh samples) indicates that drying and processing affect the level of certain phenolic acids.
Although the total phenolic acids in FD was significantly (P < 0.05) decreased, some phenolic acids did increase after being FD.These results might be due to the degradation of certain precursor phenolic acids such as gallic acid, p-hydroxybenzoic acid, p-coumaric acid and sinapic acid.Interestingly, not only chlorogenic acid, but several compounds after FD treatment also displayed a significantly higher quantity compared to fresh samples.For example, the abundance of gentisic acid, syringic acid and protocatechuic acid in FD were about 25-, 12-, and 9-fold higher than fresh samples.In SD samples, vanillin showed the highest contents compared to other treatments.Ferulic acid can be degraded into vanillin. [6]Thus, under sun-dry conditions, the increase of vanillin might come from the ferulic acid being decomposed into vanillin.Generally, among the three different dying methods, FD represented the most suitable method to preserve phenolic compounds (some compounds had even greater content than fresh samples) followed by SD and HD, respectively.
For flavonoids, 4 out of 5 compounds were identified by HPLC including rutin, quercetin, kaempferol, apigenin, and myricetin, as shown in Table 1.Kaempferol was not found in all samples.The predominant flavonoid in both fresh and dried LRL was quercetin.Among drying methods, the level of quercetin was high in FD followed by SD and much lower in HD (Table 1).Apigenin and myricetin were equally affected by all drying methods.The contents of these two flavonoids were decreased from fresh samples to a similar level in FD, HD, and SD samples.The highest content of total flavonoids was found in fresh, followed by FD, SD, and HD, respectively.Overall, FD treatment slightly affected the content of flavonoids and should be the preferred method for preserving the flavonoid content in these samples.

Changes in individual anthocyanins and carotenoids with different drying methods
According to Table 2 the effect of different drying methods on carotenoid content (b-carotene, lutein and lycopene contents) was found to be significant (P < 0.05).The carotenoid contents significantly decreased after all drying methods.The contents of carotenoids in both fresh and dried samples ranged from 47 to 307 mg/100 g db.Lycopene was found to be the predominant carotenoid in LRL fruit, followed by lutein and ß-carotene, respectively.In this study, we reported a high lycopene content in fresh samples (158 mg/100 g db).Among the different drying methods, FD provided the highest content of lycopene (116 mg/100g db), followed by SD (43 mg/100g db) and HD (20 mg/100 g db), respectively.The lycopene contents of raw tomato samples were reported in the range of 86-151 mg/100g db depending on the variety. [36]The amounts of lycopene in fresh LRL were similar to or even higher than those of tomato varieties, whereas FD showed a higher content than certain varieties of tomato. [36]Our group also previously reported a good source of lycopene in the aril oil of Thai gac (82 mg/100g db). [14]However, the lycopene contents in fresh and FD treated LRL fruit were approximately 2 and 1.4 times higher, respectively, than those of the previously reported aril oil from Thai gac.Lycopene consumption has been associated with reducing the risk of chronic diseases such as cancer and cardiovascular disease. [37]Thus, LRL fruits that are rich in lycopene could be potential sources of lycopene, enabling the development of functional foods in the future.In this study, lycopene content decreased most in HD (87%), followed by SD (73%) and FD (26%).This supports the findings of a previous study, which reported that FD and vacuum drying (VD) preserved the highest content of lycopene and b-carotene, compared to HD which had the lowest lycopene and b-carotene contents. [38]Lycopene is sensitive to heat, light, and oxygen, all of which can influence lycopene isomerization and oxidation. [39]The degradation of carotenes was observed during a long drying process, such as at 50 C with hot air dying. [38]Therefore, HD temperatures and a long drying process could lead to the greatest degradation of lycopene, whereas, FD which uses very low temperatures and vacuum conditions, preserves the highest content of lycopene compared to other drying methods.
For anthocyanin, all drying methods (FD, HD, and SD) significantly decreased the anthocyanin contents (Table 2).However, FD preserved the highest quantity of anthocyanin followed by SD and HD, respectively.Among three identified compounds (Cyanidin-3-Oglucoside, peonidin-3-O-glucoside, malvidin 3,5-diglucoside), Cyanidin-3-O-glucoside was the most predominant anthocyanin in LRL fruit.Cyanidin-3-Oglucoside is responsible for the red color in fruit and has been associated with many health benefits, such as anti-inflammatory, prevention against Helicobacter pylori infection, type 2 diabetes, cardiovascular disease and oral cancer. [40]4.Changes in organic acid contents in samples due to different drying methods The contents of 7 organic acids in LRL including oxalic acid, citric acid, tartaric acid, malic acid, quinic acid, succinic acid, and fumaric acid are shown in Table 3.The organic acids were identified based on their retention time and their content was quantified by the peak area from HPLC analysis.The results demonstrated that total organic acids content was mostly in the following order: fresh > FD > SD > HD.
The individual organic acids differed according to the drying method.Generally, FD showed the highest level for each organic acid, followed by SD and HD, respectively.However, in the case of tartaric acid and malic acid, SD displayed the highest contents, followed by FD and HD, respectively (Table 3).For all organic acids, HD showed the lowest level compared to other drying methods (Table 3).In LRL fruit, quinic acid, malic, and citric acids were the major organic acids.Quinic acid has been reported to enhance the synthesis of tryptophan and nicotinamide inducing the level of these essential compounds in humans. [32]Interestingly, quinic acid was increased by the FD method.Chlorogenic acid is the ester of caffeic acid with quinic acid. [26]There is also a proposed pathway for chlorogenic acid to be hydrolyzed into caffeic acid and quinic acid. [33]The increase in quinic acid with the FD method might result from the degradation of other compounds under the drying conditions, leading to an increase in quinic acid.In this study, chlorogenic acid was high after FD (Table 1), indicating its potential for conversion to quinic acid.Overall, in terms of organic acid content, FD represents the most suitable method to preserve or even enhance certain organic acids compared to fresh samples, followed by SD and HD, respectively.

Changes in sugar content in samples after different drying methods
Sugars are important components contributing to the sensory properties of fruits.Generally, the individual sugars found in fruits are sucrose, glucose, and fructose.These sugars are also the principal sources of perceived sweetness.Fructose was the most predominant sugar in all drying methods, followed by glucose, mannitol, sucrose, and sorbitol (Table 3).The highest fructose content was found in fresh LRL (2344 mg/g db), which is much higher than that found in Pithecellabium dulce (193 mg/g). [9]Additionally, d(þ) raffinose, d(þ) maltose, d(À) mannitol, and d(À) sorbitol were found as minor constituents in the fruits.This study identified sugar alcohols, including mannitol and sorbitol.Mannitol can be metabolized in plants. [41]The proposed pathway demonstrated that fructose 6-phosphate could be converted to mannitol and during catabolism, mannitol can also be converted to fructose 6-phosphate. [41]All drying methods reduced the contents of sucrose, glucose, and fructose, as shown in Table 3.The stability of sugars is influenced by various factors including temperature and pH. [42,43]Several studies have reported the decreased in glucose and fructose levels following drying process. [44,45]For instance, fructose content decreased by approximately 23% and glucose levels were reduced by around 11% under the hot drying process (60 C). [45] Another study also reported a reduction in glucose levels with hot air drying compared to freeze drying. [44]However, HD and FD increased the mannitol content by 13 and 11 times, respectively, compared to fresh samples (Table 3).The possible explanation for the increase in mannitol content is that the rise in temperature during the sample preparation and the progress of FD and HD lead to the activation of enzymes that stimulate the metabolism of macromolecule sugars. [46]Whilst, the mannitol content of SD samples slightly decreased compared to the fresh samples in our present study.Fructose can be hydrogenated to mannitol [47] as shown in the pathway presented in Supplementary data (Figure S1).Therefore, the high content of mannitol in HD might be attributed to the conversion of fructose into mannitol.

Relationship between phenolic compounds and sugar content degradation as analyzed by FTIR spectroscopy
In this study, we observed the degradation of sugars with different drying methods.Figure 2 displays the FTIR spectroscopy spectra of LRL samples affected by various drying methods compared to the fresh samples.Each peak corresponds to the absorption of functional groups present in the samples.Fructose, one of the most abundant sugars naturally found in fruits, can undergo degradation to form mannitol. [47] Mannitol was characterized by functional groups, including O single-bond H stretching (3350-3450 cm À1 ) and C single-bond H stretching (2850-2950 cm À1 ). [48]The characteristic peaks of mannitol were identified at 3300 cm À1 , 2933 cm À1 , and 2855 cm À1 .These peaks showed high intensity in HD and FD samples, whereas they revealed low intensity in fresh and SD samples (Figure 2).The FTIR spectroscopy data support the high content of mannitol in HD and FD, as opposed to the low amount in SD and fresh samples (Table 2).Fructose can be hydrogenated to mannitol, [47] as explained in the pathway shown in Supplementary data Figure S1.Factors such as temperature and time could influence hydrogenation efficiency.For instance elevating the temperature from 60 C to 90 C results in an improved yield of hexitols (sorbitol and mannitol). [47,49]

Conclusion
According to the results obtained from this study, LRL has been considered a potential source of bioactive compounds, particularly lycopene, cyanidin-3-O-glucoside, and quinic acid.It can be seen obviously that different drying methods have a significant impact on the phytochemicals present in LRL, including phenolic acids, flavonoids, carotenoids, anthocyanins, organic acids, and sugars.Overall, FD is a suitable method to preserve these compounds as well as their antioxidant activities, except for DPPH values.However, mannitol content was found to be enhanced by HD and FD methods.This present study has provided valuable information for the further development of functional ingredients or products derived from LRL.

Table 1 .
Contents of phenolic acids and flavonoids in Lepisanthes rubiginosa (Roxb.)Leenh with different drying methods.Values are expressed as mean ± SD of triplicate measurements (n ¼ 3).ND: Not detected; FD: Freeze drying; HD: Hot air drying; SD: Sun drying.Means with different letters (a, b, c, d) are significantly different at P < 0.05 within the same column in the parameter.Means with different letters (A, B, C, D) are significantly different at P < 0.05 within the same row in the parameter.

Table 2 .
Contents of carotenoids and anthocyanins in Lepisanthes rubiginosa (Roxb.)Leenh with different drying methods.Values are expressed as mean ± SD of triplicate measurements (n ¼ 3).ND: Not detected; FD: Freeze drying; HD: Hot air drying; SD: Sun drying.Means with different letters (a, b, c, d) are significantly different at P < 0.05 within the same column in the parameter.Means with different letters (A, B, C, D) are significantly different at P < 0.05 within the same row in the parameter.

Table 3 .
Contents of organic acids and sugars in Lepisanthes rubiginosa (Roxb.)Leenh with different drying methods.Values are expressed as mean ± SD of triplicate measurements (n ¼ 3).ND: Not detected; FD: Freeze drying; HD: Hot air drying; SD: Sun drying.Means with different letters (a, b, c, d) are significantly different at P < 0.05 within the same column in the parameter.Means with different letters (A, B, C, D) are significantly different at P < 0.05 within the same row in the parameter.