Enhancement of Phenolics Content and Biological Activities of Longan (Dimocarpus Longan Lour.) Treated with Thermal and Aging Process


 This study is the first to successfully produce novel black Dimocarpus longan by undergoing thermal and ageing process. Pericarp, aril, and seed of black D. longan were macerated in 95% v/v ethanol. Their chemical compositions were investigated by Folin–Ciocalteu assay, aluminium chloride assay, and high-performance liquid chromatography. Antioxidant activities were evaluated in terms of radical scavenging and iron (III) reducing capacity. Enzyme inhibitory assay was used to evaluate the hyaluronidase inhibition. Black D. longan seed extract contained the significantly highest content of flavonoids and phenolic compounds (p < 0.05). Each gram of the extract contained 53.6 ± 0.9 mg of gallic acid, 19.8 ± 2.9 mg of corilagin, and 24.5 ± 0.7 mg of ellagic acid. This extract was the most potent anti-hyaluronidase and antioxidant with the strongest free radical scavenging activity and reducing power. Therefore, it was proposed as functional food and further used in the pharmaceutical or cosmeceutical industries.


Introduction
Dimocarpus longan Lour., a subtropical evergreen plant in the family of Sapindaceae, is widely known as longan. D. longan can be cultivated in several countries in East Asia, South-East Asia, Australia, and some subtropical regions in the US. 1 China and Thailand are the largest area of commercial D. longan cultivation. 2 The succulent and edible aril with delicious avor and health bene ts lead D. longan to be more and more popular. 1 Since the aril part of D. longan contains several polyphenols, avonoids, organic acids, and polysaccharides, it possessed various bene cial biological activities, including antioxidant, antiglycation, anticancer, immunomodulatory, prebiotic, anti-osteoporotic, anxiolytic, and memoryenhancing effects. 3 A decoction of the dried aril has been taken as a tonic for insomnia and neurasthenic neurosis treatment since an ancient time. 4 Not only the aril part of D. longan, which is the only edible portion, that has been reported for the health-bene cial effects, the pericarp has also been reported to contain abundant polyphenols, avonoids, and polysaccharides, which possessed antioxidant, antityrosinase, and anti-hyperglycaemic activities. 1 On the other hand, D. longan seed, which is a waste from the food and canning industry, contains antioxidative polyphenols and possesses anti-tyrosinase, antibacterial, and anti-fungal activity. 1,5,6,7 Besides, D. longan seed has been administered to counteract heavy sweating, whereas, the pulverized kernel was used for astyptic treatment according to the components of saponin, tannin, and fat . 8 Although D. longan have been reported to contain variety of biological active components and have a potential to be used for the treatment of various conditions, the fruit of D. longan has a short storage life since its pericarp rapidly turns brown and harden at ambient temperature. 1,9 Even if low temperature (1-5°C) could preserve D. longan from pathological decay, the fruit deteriorate easily after remove from the cold storage. 3 Therefore, dried esh and the canned product of D. longan are widely consumed and can be distributed worldwide. Generally, dried foods can be kept for a longer period, but their sensory and nutritional characteristics are often lost along with the water removal during the drying processes. 10 Production of intermediate moisture food (IMF) is another technique to overwhelm this problem since the properties of IMF are close to fresh foods yet having extended the shelf life. 10,11 A reduction of the moisture content and a water activity below 0.6 will not support the microbial growth and leads to shelf-stable products. 11,12 Various types of food have been preserved as IMF, such as meat and several fruits, e.g. grapes, tomatoes, peaches, prunes, apricots, strawberries, etc. 10,13 However, some additives are required in the production of IMF for antimicrobial (e.g. preservatives, sugar, and salt), along with the agents for water activity reducing and plasticizing, e.g. glycols and sorbitol. 11,12 Beside IMF, the heating and ageing process could also prolong the shelf life without refrigeration. A well-known food undergone this process is black garlic (Allium sativum L.), a processed garlic produced by thermal treatment on raw garlic at high temperature and high relative humidity for 60-90 days without using additives. 14,15 During the production process, raw garlic also undergoes Maillard reaction, which occurs between amine groups and carbonyl compounds, nally resulting in brownish melanoidin. 16 A variety of biological activities of melanoidin has been demonstrated, including antioxidant, antibacterial, anti-in ammatory, hypoglycemic, hypotensive, antitumor effects, prevention of obesity, lowering of serum lipopolysaccharide levels, and modulation of the composition of the gut microbiota. 16,17 Additionally, inhibition against oxidation and angiotensin I converting enzyme were enhanced in black garlic comparing to raw garlic. 15 Therefore, the heating and ageing process not only preserved the food but also enhanced their biological activities. Since the production of black D. longan undergone heating and ageing process has not been reported before, this study is the rst to produce a novel black D. longan and investigate the chemical compositions, as well as its health bene t potential in antioxidant, anti-in ammatory, and anti-hyaluronidase activities.

Results And Discussion
DriedD. longanand blackD. longanextracts The external appreance of dried D. longan were totally different from black D. longan as shown in Fig. 1. The color of pericarp, aril, and seed of black D. longan was obviously darker than that from dried D. longan, especially the aril part which turn from dark brownish color to black color. The pericarp and seed of black D. longan were substantially more moist than dried D. longan. The outer part of dried D. longan seed was shriveled, whereas, the dried D. longan pericarp was dry and brittle. All D. longan extracts were semisolid mass with different color as shown in Fig. 1. The color of black D. longan extracts were darker than the dried D. longan extracts. The color of pericarp extracts were the darkest, followed by the extract from seed and aril, respectively. Yields of each D. longan extracts are shown in Fig. 2. The aril part yielded the highest extract content, followed by pericarp and seed, respectively. The highest yield of the extract was obtained from black D. longan aril (21.6% w/w), followed by dried D. longan aril (17.6% w/w), dried D. longan pericarp (13.8% w/w), black D. longan aril (11.0% w/w), black D. longan seed (6.6% w/w), and dried D. longan seed (3.6% w/w), respectively.
Chemical compositions of driedD. longanand blackD. longanextracts D. longan extracts were investigated for the content of total phenolic compounds, total avonoid, gallic acid, corilagin, and ellagic acid. Gallic acid and corilagin are natural polyphenolic compounds which belong to hydrolyzable tannin, whereas, ellagic acid belongs to a avonoid group. 18 Among different parts of dried D. longan, pericarp extracts contained the signi cantly highest total phenolic content (p < 0.05) and the highest total avonoid as shown in Fig. 3. The results were in a great agreement with the previous study reported that polyphenolic compounds are abundant in pericarp and seed of D. longan compared to the D. longan aril. 1 The total phenolic content of pericarp, seed, and aril extracts from dried D. longan, which were 967.6 ± 31.5, 739.3 ± 62.3, and 229.5 ± 2.6 µg GAE per g extracts, were found in agreement with the previous study reported that the total phenolic content of D. longan were in the range of 22.09-132.47 mg of gallic acid equivalents (GAE/100 g), which were equivalent to 220.9-1,324.7 µg GAE per g extracts. 1 Interestingly, the dramatically enhanced in total phenolic content was observed in black D. longan seed extract. The ethanolic extract from black D. longan seed contained as high as 1827.1 ± 73.1 µg GAE per g extracts, which was much higher that the previous reported. 1 On the other hand, there was no signi cantly difference between the total phenolic content of dried and black D. longan extract from pericarp and aril (p > 0.05).
In addition to the total phenolic content, black D. longan seed extract also contained the signi cantly highest avonoids content (p < 0.05). Among various dried D. longan extracts, the pericarp part contained the signi cantly highest avonoid content of 2.8 ± 2.4 µg QE per g extract as shown in Fig. 3 (p < 0.05). The results were in a great accordance with the previous study reported that the quercetin content of D. longan pericarp were 3.12 ± 0.76 mg/kg, which was equivalent to 3.12 ± 0.76 µg per g extract. 19 Besides, D. longan pericarp has been reported to contain slightly higher content of avonoids than D. longan seed and aril. 20 Obviously, the avonoid content of black D. longan seed extract, which was as high as 13.6 ± 2.5 µg QE per g extract, was dramatically enhanced, which was about four times higher than previously reported.
Although the thermal and ageing process of D. longan did not affect the phenolic and avonoid content of D. longan pericarp and aril, the total phenolic and avonoid content of D. longan seed were obviously enhanced after the production process of black D. longan. The likely explanations might be due to the formation of biological compounds, which were not originally present in the D. longan seed, during the thermal and ageing process. 21 Gallic acid, corilagin, and ellagic acid have been identi ed as major polyphenolic components of D.
longan pericarp and seed in several previous studies. 22,23,24 Therefore, the content of these polyphenolic components were also investigated in black D. longan extract, in a comparison with dried D. longan extracts. The HPLC chromatograms of gallic acid, corilagin, and ellagic acid are shown in Fig. 4. The peak of gallic acid, corilagin, and ellagic acid were detected at around 3.7, 9.9, and 19.2 min, respectively. The content of gallic acid, corilagin, and ellagic acid of each D. longan extracts are shown in Fig. 5. The results were in a good agreement with their total phenolic and total avonoid content since black D. longan seed extract contained the signi cantly highest polyphenolic compounds and avonoids content of (p < 0.05).
Among various parts of dried D. longan fruit, seeds contained the signi cantly highest content of gallic acid, corilagin, and ellagic acid (p < 0.05) with the amount of 5.3 ± 0.0, 8.9 ± 0.1 and 1.9 ± 0.2 mg/g extract, respectively. Interestingly, the content of these phenolic and avonoid were signi cantly enhnaced after the production process of black D. longan (p < 0.05). The gallic acid, corilagin, and ellagic acid content of black D. longan seed extract were as high as 53.6 ± 0.9, 19.8 ± 2.9, and 24.5 ± 0.7 mg/g extract, respectively. The content of gallic acid and ellagic acid were about ten times increased, whereas, the content of corilagin was double when the D. longan undergone thermal and ageing process. The reason might be due to the liberation of free polyphenolic and avonoid from the bound forms (i.e. esteri ed and glycosylate) or the decline in enzymatic oxidation involving in the antioxidant compounds in the raw fruit. 25 The results of black D. longan were in accordance with the black garlic as the total phenolic and total avonoid content of the garlic subjected to the thermal processing steps were signi cantly higher than those of fresh garlic. 21,25 The previous study reported that the phenolic content was increased by about 4-10-fold in the black garlic cloves compared with the fresh garlic. 21 Antioxidant activities of driedD. longanand blackD. longanextracts The antioxidant activities of dried and black D. longan extracts were investigated by two assays with different mechanisms of action. The ABTS assays referred to the electron transfer reaction and represented the radical scavenging activity of the tested samples, while the FRAP assay referred to the ion reduction process, which represented the ability of the tested compound to convert ferric ions (Fe3 + ) to ferrous ions (Fe2 + ). 26,27,28 The ferric reducing antioxidant power (EC 1 ) and TEAC value of dried and black D. longan extracts are shown in Fig. 6.
The TEAC value of black D. longan extracts were not signi cantly different from dried D. longan extracts, except in the aril part. The dried D. longan aril extract had no antioxidant activity, whereas, the black D. longan aril extract possessed some antioxidant activity with the TEAC value of 4.1 ± 1.4 µg Trolox/mg extract. The probable explanation lies in the greater Maillard reaction which occurs in the aril part as compared with the others. As Maillard reactions refer to a chemical reaction between an amino acid and a reducing sugar in the presence of heat 29 , D. longan aril, which contained glucose, fructose, and various types of amino acids, e.g. γ-aminobutyric acid, tended to undergone Maillard reaction. 2 Besides, this nonenzymatic browning reactions led black D. longan a darker color and resulting in the formation of some antioxidant compounds. 25 On the other hand, black D. longan pericarp and seed extracts possessed the same radical scavenging activity as that from dried D. longan. The likely possibility might be because of the degradation of some oxidative compounds during the heating process although some free polyphenolic and avonoid were liberated from the bound forms. 21 Interestingly, the TEAC values of pericarp and seed extracts from both dried and black D. longan were comparable to ascorbic acid, gallic acid, and corilagin (p > 0.05). Ellagic acid was remarked as the most potent radical scavenger (TEAC = 23.4 ± 0.3 µg Trolox/mg), followed by ascorbic acid (TEAC = 12.3 ± 0.0 µg Trolox/mg), gallic acid (TEAC = 12.8 ± 0.2 µg Trolox/mg), and corilagin (TEAC = 12.7 ± 0.1 µg Trolox/mg). Thereby, ellagic acid was found to be the main compound responsible for the free radical scavenging activity of D. longan extracts together with gallic acid and corilagin. 30 Although the previous study reported that among various polyphenolic compounds, tannins demonstrated the strongest ABTS•+ radical scavenging activity 31 , the present study remarked that ellagic acid, which belongs to a avonoid group, was more potent than gallic acid and corilagin, which belong to hydrolyzable tannin. 1 Furthermore, D. longan extracts from both pericarp and seed part could be hence remarked as the natural extracts with potent radical scavenging activity.
Aside from radical scavenging activity, D. longan extracts also possessed a reducing ability as shown in Fig. 6. The reducing ability of D. longan extracts were in a very well accordance with their phenolic and avonoid content. Gallic acid possessed the signi cantly highest EC 1 value of 237.0 ± 1.6 mM FeSO 4 /mg, which was comparable to that of ascorbic acid (238.3 ± 0.2 mM FeSO 4 /mg), followed by corilagin (226.2 ± 2.9 mM FeSO 4 /mg) and ellagic acid (192.3 ± 0.7 mM FeSO 4 /mg), respectively. However, both phenolic and avonoid were responsible for their reducing capacity. 28 The black D. longan seed extract, which contained the highest content of total phenolic, total avonoid, gallic acid, corilagin, and ellagic acid, thus possessed the signi cantly highest reducing ability with the EC 1 value of 150.0 ± 1.0 mM FeSO 4 /mg extract (p < 0.05). Consequently, the black D. longan seed extract was suggested as the most potent antioxidant extract with the strongest free radical scavenging and reducing ability.
Anti-in ammatory activities of driedD. longanand blackD. longanextracts The inhibitory activities against the secretion of IL-6 and TNF-α, which are key players involved in the agerelated in ammatory process 32 , of dried and black D. longan extracts were investigated and the results are shown in Fig. 7. TNF-α is known as an indicator of chronic in ammatory processes related to ageing, whereas, IL-6 has been noted as the cytokine for geriatricians, which are associated with poor physical performance, muscle weakness, and could predict the onset of disability. 33,34 Among various parts of D. longan fruit, aril of both dried and black D. longan were predominant in the IL-6 and TNF-α inhibition. Gallic acid was suggested to be the main compound responsible for both IL-6 and TNF-α inhibitory activities. In contrast, corilagin was responsible only for TNF-α inhibition. Although D. longan extracts and their major chemical components exhibited only low to moderate anti-in ammatory activities comparing to dexamethasone, a corticosteroid used in the treatment of in ammations, they were suggested as natural anti-in ammatory products with no steroidal side effect.
Anti-hyaluronidase activities of driedD. longanand blackD. longanextracts Hyaluronidase, a homologous enzyme that hydrolysis or depolymerise hyaluronan, plays an important role in the modulating activity of many pathological processes. 35 Hyaluronan plays a pivotal role in the maintenance of the elastoviscosity of liquid connective tissues and control the water transportation that was related to the tissue hydration. 36 The degradation of hyaluronan resulting in the production of breakdown products, which are the signal that injury has occurred and initiates an in ammatory response. 35 Additionally, hyaluronan has been known an lubricant and shock-absorber in joints and connective tissues. 37 Its degradation hence leads to the deterioration of viscoelastic properties of the synovial uid. 38 The inhibitory activities against hyaluronidase of dried and black D. longan extracts are shown in Fig. 8. Although D. longan extracts exhibited low anti-hyaluronidase actvitiy, the inhibitory effect of black D. longan seed was signi cantly enhanced compared to the dried D. longan seed extract. Since the antihyaluronidase activity of black D. longan seed extract (18.4 ± 2.0%) was the most signi cantly potent (p < 0.05), black D. longan seed extract could be suggested for the anti-hyaluronidase apart from its antioxidant activities.
In conclusion, Black D. longan was successfully produced after undergone thermal and ageing process in the controlled temperature of 70° C and controlled humidity of 75% RH. The external appearance of black D. longan was totally different from that of dried D. longan. The ethanolic extract of black D. longan seed contained the most signi cantly abundant of total phenolic, total avonoid, gallic acid, corilagin, and ellagic acid content (p < 0.05). Furthermore, the black D. longan seed extract possessed the most signi cantly potent antioxidant and anti-hyaluronidase activities. The radical scavenging activity, reducing power, and anti-hyaluronidase activities of black D. longan seed extract were obviously higher than that of dried D. longan seed extract and the previously reports. Therefore, black D. longan seed extract was suggested for further used topically for the promotion of various health bene ts, such as antiskin ageing, joint pain reliever, etc. On the other hand, the aril part of both dried and black D. longan possessed signi cant anti-in ammatory activities. They were suggested as natural edible antiin ammatory agents. Each D. longan extract was analyzed for total phenolic content using the Folin-Ciocalteu method according to the previously described method. 40 The results were presented in the form of gallic acid equivalent values (GAE) representing an amount of gallic acid (µg) per g of the D. longan extracts. GAE was calculated following the equation; X = (Y -0.0075)/0.3812, where X is GAE or µg of gallic acid per g of the D. longan extracts and Y is an absorbance of each sample tested in Folin-Ciocalteu assay. The experiments were triplicately performed.

Total avonoid content determination
Total avonoid content of each D. longan extracts was investigated using aluminium chloride method which has been previously described with some modi cations. 41  Anti-in ammatory activities determination of dried D. longan and black D. longan extracts Murine monocyte-macrophage (RAW 264.7) cells (American Type Culture Collection, ATCCTIB-71) treated with LPS were used to investigate the effect of D. longan extracts and their chemical compositions on the pro-in ammatory cytokine secretion (IL-6 and TNF-). The cell culture was performed according to a method previously described with some modi cations. 43,44 The treated cells along with its supernatant were divided into two parts. The rst withdrawal part was undergone centrifugation for 10 min at 13,500ǵ and the supernatant was investigated for the cytokine secretion by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's protocol (R&D Systems). On the other hand, the second part, which was leftover in the wells, was investigated for the cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. To reduce variation due to cell density differences, secretion of IL-6 and TNF-α from RAW 264.7 cells were normalized to MTT levels. 44