The Identification of Phytocompounds from Limonia acidissima L. Fruit: A Short Report and Updated Review-Focusing on Antihyperuricemic and Antiarthritic Activities

ABSTRACT


INTRODUCTION
Hyperuricemia is joint inflammation due to the accumulation and deposition of monosodium urate which can cause various signs and symptoms such as pain, redness, swelling and joint stiffness [1].Hyperuricemia is a major risk factor for gouty arthritis [2] .The use of natural ingredients as alternative medicine for metabolic diseases has been massively campaigned by the government of the Republic of Indonesia through the slogan "back to nature".The agenda of this program include obtaining new bioactive compounds as well as to reveal the role of nutrients contained in plants in overcoming public health problems.Our research group has an experience in responding this call [3]- [5].Limonia acidissima Linn (L.acidissima) is a plant originating from India and Sri Lanka and then spread to Indonesia.So far, it has been used to treat diarrhea and dysentery [6], diabetes [7], wound healing and antioxidants [8], hepatoprotective [9] and as a cosmetic ingredient [10].Preliminary results of our research show that NIRS (Near-infrared spectroscopy) results of L. acidissima fruit from five districts in Aceh, Indonesia identified the presence of CH, OH, CHO and NH molecular bonds as the main components forming organic matter.This is related to the carbohydrate, acid, protein or fiber content of this fruit [11].In this present work, we extracted L. acidissima fruit using sequential maceration using n-hexane, ethyl acetate, ethanol, and methanol solvents (non-polar to polar).Further, the extracts were analyzed using Gas Chromatography -Mass Spectroscopy (GC-MS) to reveal their phytoconstituents.To accompany the analysis results, we presented the updated literature review.This report is a part of project that aims to discover the antihyperuricemic and antiarthritic potentials of Limonia acidissima Linn fruit.
Prior to extraction, fruits of L. acidissima (12 kg) were separated from its peels and seeds, and subsequently oven-dried at 40°C.The sample was crushed into find powder.The maceration was performed on simplicial powder (2 kg) in a sealed container using n-hexane and sequentially partitioned using ethyl acetate, ethanol, and methanol.The maceration was repeated 3x24 h, while stirred occasionally.In each repetition, the fil-trate was separated using filter paper and concentrated with rotary evaporator (40°C; 30 rpm).This process yielded extract samples as much as 4.3 g (0.215%), 75 g (3.75%), 110.8 g (5.52%), and 55.6 g (2.78%) for n-hexane, ethyl acetate, ethanol, and methanol, respectively.Each extract sample was stored in a sealed container at 4°C until further use.

Phytocompounds Identified
The phytochemical screening of L. acidissima fruit revealed the presence of flavonoids, polyphenols, alkaloids, tannins, saponins, and the absence of alkaloids mayer (Table 1).Flavonoids are phytochemical products known as polyphenols which are found in many herbs and plants, they are important anti-oxidative agents in counteracting free radicals.However, in the human digestive system, absorption of flavonoids in the intestine is very low and excretion is rapid and abundant through the process of urine excretion [12].Terpenoids are the main components of essential oils which have been reported as anticancer, antimicrobial, antiinflammatory, antioxidant and anti-allergic [13].Tannin is a secondary metabolite of phenolic compounds which causes a bitter and astringent taste in L. acidissima fruit.It can react and coagulate proteins or other organic compounds containing amino acids and alkaloids.
Previous research results reported various potential benefits of L. acidissima for health due to its high phenolic content.L. acidissima fruit extract was studied to have antioxidant properties by increasing the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) and reducing lipid peroxidation in serum, liver and muscle tissue.Anti-inflammatory activity of methanol extract of L. acidissima by inhibiting carrageenan-induced paw edema and cotton pellet granuloma formation in animal models [14].
Results from GC-MS analysis on L. acidissima fruit extracts obtained using n-hexane, ethyl acetate, ethanol, and methanol as solvents, respectively, have been presented in Table 2. GC-MS analysis revealed the dominant presence of fatty acids in n-hexane and ethyl acetate extracts of L. acidissima fruit.The contents of oleic acid, n-hexadecanoic acid, palmitoleic acid, and octadecanoic acid were dominant in n-hexane and ethyl acetate extracts.Sterol groups such as γsitosterol, stigmasterol, ergost-5-en-3-ol, (3beta.)were identified in the ethyl acetate and ethanol extracts, but both had larger peak areas in the latter.As for the methanol extract, its content was rich in 2-pentanone, 4-hydroxy-4-methyl-, oleic acid and 9-octadecenoic acid (Z)-methyl ester.Previously, five main fatty acids have been identified in L. acidissima, namely palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid [9].Monounsaturated fatty acids such as oleic acid are considered effective in lowering lowdensity lipoprotein (LDL) cholesterol levels thereby reducing the risk of coronary heart disease and preventing insulin resistance [9].Oleic acid exhibits analgesic and anti-inflammatory activity by downregulating the expression of proinflammatory cytokines such as tumor necrotic factor alpha (TNF-α), interleukin 6 (IL-6) and nitric oxide (NO), as well as the production of free radicals in macrophage-stimulated lipopolysaccharide cells.Meanwhile, polyunsaturated fatty acids could reduce pro-inflammatory mediators or downregulate inflammatory cytokines such as IL-6, TNF-α, and monocyte chemotactic protein-1 (MCP-1) [15].The compound n-hexadecanoic acid has been considered to have antioxidant and antiinflammatory activities [16], [17] as well as an inhibiting activity against xanthine oxidase (XO) [18].
The results of previous in vitro studies have shown that triterpenoids exhibit potential anti-UA effects through their anti-inflammatory effects, while tannins exhibit a dual effects through UA formation inhibition and uricosuric action [19].In this present study, the presence of triterpenoids and related compounds present illustrates the extract potentials as an antiinflammatory and antioxidant which are important in treating hyperuricemia and gouty arthritis.Antioxidants prevent the oxidation of molecules in cells and protecting healthy cells from damage caused by free radicals and unstable and highly reactive molecules produced as by-products of uric acid metabolism.

Updated Literature Review
Sitosterol and its glycosides and stigmasterol are reported to have anti-inflammatory and immune-modulating activities.Although sitosterol failed to reduce serum UA levels in hyperuricemic rats, it was able to reduce leg swelling caused by monosodium urate crystals [20].Based on the literature search, fatty acids along with other small phytocompounds are potential for treating gout, where the data have been summarized in Table 2. Previous studies have proven the activity of several plants belonging to the same family as L. acidissima (rutaceae).Fruit juice and lemon extract significantly decreased serum uric acid levels in human and rat subjects.The underlying mechanism suggests that lemon may lower serum uric acid independent of xanthine oxidase inhibition [29].Apple juice intake has been shown to reduce plasma uric acid in fructose-induced model [30].Oranges, grapefruit, and lemon juice were

Table 1 .
Qualitative phytochemical screening of L. acidissima extract

Table 2 .
Summary of L. acidissima compounds with various extraction methods and their activities