The metabolomic profile of Garcinia intermedia leaves with several sample drying methods

Garcinia intermedia, commonly called cherry mangosteen or lemon drop mangosteen, originates from Mexico and Central America. This plant is not cultivated in Indonesia because the fruit is less tasty than mangosteen (Garcinia mangostana). Information about the metabolome content in Garcinia intermedia needs to be known to increase its commercial use. Several studies have shown that this species has anti-microbial content that benefits health. This study aims to perform metabolomics profiling in the leaves of Garcinia intermedia using 3 (three) treatments of leaves drying methods such as air-dried (room temperature), freeze-drying, and oven (hot air) through Gas Chromatography-Mass Spectrophotometry (GC-MS). The results identified a total of 35 different compounds in the leaves of Garcinia intermedia. The highest area pct was Friedelan-3-one (29.92%) obtained from air-dried preparation method. The other identified compounds were the sesquiterpenes group (beta-Caryophyllene, gamma-Muurolene, Naphthalene,1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1- (1-methylethyl)-,(1S-cis)-, and 1-Isopropyl-4,7-dimethyl-1,2,3,5,6,8a-hexahydronaphthalene), Alkane group (Octadecane and Heptacosane) and vitamin E (delta-Tocopherol and dl-alpha-Tocopherol). This study revealed the insight of potential uses of Garcinia intermedia leaves extract with additional functional metabolites that could further be used to discover potential drugs that benefits for human health.


Introduction
Garcinia intermedia (Pittier) Hammel. is not a popular fruit compared to other Garcinia species.This species is native to Mexico and Central America and is also found in Ecuador and Columbia [1].This plant has a wide area of adaptation from the lowlands to the highlands with an altitude of 1000 meters above sea level.In addition, it is also shade tolerant and resistant to marginal soil stress.The propagation of this plant uses seeds.This species is also cultivated in Australia.In Indonesia, it is an introduced plant and can be found in the genetic resource collection gardens of the Tropical Fruit Plants Research Institute in West Sumatra.In Indonesia, this plant is not cultivated commercially because the plant is not yet of high economic value.This species has round fruit, green young skin color and yellow ripe fruit.The fruit weight is 71-140 g with an edible portion of about 52%, the rest are seeds and skin, the taste of fresh sweet and sour fruit flesh.In Zirimicuaro maxico, this fruit has a higher consumption value than other fruits [2].Several studies have shown that G. Intermedia fruit has medicinal properties because the benzophenone extracted from the fruit inhibits the growth of human colon cancer cells [3].The leaves of this plant are green and have a thick cuticle on the abaxial side.Abe at.al. [4] conducted research on trypanocidal in G. intermedia leaves.The results show that Guttiferone A in the leaves can inhibit the attack of the blood parasite that causes surra, namely Trypanosoma evansi.This discovery suggests that the leaves of this plant function as an antiparasitic.Information about the benefits of G. intermedia for human and animal health needs to be continuously developed to increase the economic value of this plant and make it is suitable for cultivation.It should be noted that the metabolome content in the leaves of G. Intermedia.The application of metabolomics technology in the field of biology includes informative analysis to characterize and identify compounds of interest.Metabolite profiling (analysis without a target) can offer a comprehensive characterization of sample properties and enable the identification of new compounds related to the characteristics of the sample without bias [5].
One of the techniques used in metabolomics profiling is Gas Chromatography Mass Spectroscopy (GC-MS).GC-MS is used to analyze mixtures of volatile compounds or compounds with volatile and heat-stable derivatives.GC-MS has high chromatographic conversion power, a robust quantification method, and the ability to identify high-fidelity metabolites [6].Derivatization is required for the analysis of certain compounds to increase their volatility and thermal stability.Gas chromatographymass spectrometry (GC-MS) based metabolomics is ideal for identifying and quantifying small molecules (<650 Da).Compounds that can be analyzed by this method include organic acids, amino acids, sugars, aromatic amines, fatty acids, and so on [7].GC-MS has a high level of sensitivity and precision for detecting compounds.The disadvantages of this method are its limited use for analyzing volatile compounds and the need for derivatization for certain compounds.This study aims to profile the metabolites in the leaves of G. intermedia using the GC MS method.

Preparation and drying treatment
The material used leaves of Garcinia intermedia from Indonesian Tropical Fruit Research Institute at West Sumatera Indonesia.It was washed with tap water to remove debris.The drying method consists of three different methods following: (I) air-dried, sample stored at room temperature (20 ± 5 •C) for eleven days); (II) drying in a hot air oven at 40 o C, sample stored in the oven at 40 o C for three days; (III) freeze-drying method, using freeze-drying from Snijders scientific (-22 o C, vacuum pressure of 0.001 millibars) for two days.All powder samples were stored at 4 o C until further use.The dried leaves materials were blended into powder using an electric blender.One gram of powder was weighed and transferred to the Soxhlet apparatus for extraction, where the pulverized powder was extracted exhaustively with 99% ethanol.The final residue thus obtained was then subjected to GC-MS analysis.

The metabolomic profile of Garcinia intermedia leaves
The Garcinia genus consist of species with many beneficial functional metabolites.The little-known species, G. intermedia, has been consumed as fresh fruit in their natural origin, the tropical area of Mexico and Central America.The Mexican and Central American people consume for its nutritional and medicinal value such as the fruit extract benzophenones that could inhibit the human colon cancer growth [8].The metabolomic study has been highly developed nowadays.The study focuses on analyzing the potential of phytochemical compounds from natural sources such as fruits, leaves, or roots of the plant using a gas chromatography and mass spectrometry (GC -MS) system.This technique is highly reliable in separating and identifying the volatile and semi volatile compounds in their pure form and gives more metabolomes product from natural resource [9].In this study, the GC -MS was used to analyze the metabolomic profile on leaves of G. intermedia that revealed a total of 124 peaks (Figure 1) with 35 different organic compounds identified (Table 1).The 35 different volatile/semi volatile organic compounds with the quality of similarity percentage ≥ 70% were produced by GC -MS analysis of Garcinia intermedia leaves out of 60 total identified compounds.The G. intermedia metabolomes majority consist of the terpenes class.The terpenes have the sesquiterpenes group (C15H24) triterpenoid group (C30H50) and about 50% out of all the identified compounds.The other compound groups are vitamin E (tocotrienol and tocopherol), alkane, organochlorine, and alcohol.The alcohol/the compound groups?were also found in the leaves of G. intermedia.The ten highest GC -MS area peak percentages (area pct) of the compounds from each method are listed in Figure 2.  Different drying methods were applied to the leaves sample preparation.Based on the drying methods, the metabolomic profile showed the compound's different types and peak areas.The number of peaks 46, 43, and 32 (Figure 1) were identified from the leaves of G. intermedia using the air-dried, freeze, and oven (hot air) drying preparation methods, respectively.The drying method could decrease the amount of many volatile compounds such as terpenes, sesquiterpenes, and alcohols due to the compound degradation or the evaporation of such compounds by water removing with too high or low temperature during the process [10].The air-dried methods produced more amount and number of identified compounds than other drying methods.The highest compound area peak percentage (area pct) was produced from the treatment of air-dried method at 29.92% and identified as Fridelan-3-one (Friedelin).The amounts of this compound were decreased in other drying methods of freeze-drying (15.10%) and oven drying (17.87%).The volatiles characteristic of fresh guava fruit also decreased by the process of oven and freeze-drying, but oven drying could promote the insoluble flavonoids such as quercetin [11].The convective and natural drying method using air-dried or sun drying is ideal for producing the highest content of volatile compounds and intensity of critical attributes of dried Boletus edulis [12].The freeze-drying method gives the best performance to promote beta-Caryophyllene and vitamin E content in the metabolomes.The compound produces better area pct of beta-Caryophyllene (8.18%), delta-Tocopherol (4.73%) and dl-alpha-Tocopherol (3.90%) compared to the oven and airdried.The different approaches of drying methods for the leaf extract preparation process could be delivered a specific profile of different compounds with more intensity and availability in the extracts.
The Friedelan-3-one, as one of the significant constituents of G. intermedia leaves metabolomes, produces the highest peak out of all detected compounds.Like the sesquiterpenes group, this compound is a member triterpenoid group of terpenes class.Friedelan-3-one has been found in several plant extract and investigated as a potential drug candidate, antimicrobial agents, and cosmetics application.It is the first report of the isolated Friedelan-3-one compound from leaves of Garcinia intermedia using Soxhlet apparatus and 99% ethanol extraction by GC -MS analysis system.The Friedelan-3-one was successfully isolated from other Gracinia genus.They are from the leaves of G. multiflora [19] and G. hombroniana Pierre that had potential uses for Alzheimer's disease inhibition [20].The summary of other potential uses of Friedelan-3-one listed in Table 2.
This study first reports the preliminary metabolomic profile on leaves of G. intermedia using the GC -MS system.This result could give an insight into potential uses of G. intermedia leaves extract that consists of the functional metabolites could be further used to discover potential drugs that benefits for human health.

Conclusion
The metabolomic profile of Garcinia intermedia leaves extract by GC -MS system identified 35 different compounds.The triterpenoid Friedelan-3-one showed the highest peak area (area pct) with 29.92% that obtained from air-dried preparation method.The other identified compounds were the sesquiterpenes group, Alkane group, and vitamin E that mostly found in all drying process treatment.This study revealed the insight of potential uses of G. intermedia leaves extract that consist with the functional metabolites that could benefit human health as a potential drug agent.

Contributorship
All the authors approved the final manuscript and contributed as main (MAC) and minor (MIC) contributors.Tri Budiyanti (MAC), designed, collected the samples, carried out the experiment, analyze the data and write the manuscript; Sri Hadiati (MAC), Jumjunidang (MIC), Ellina Mansyah (MIC) NLP.Indriyani (MIC), W. Nawfetrias (MIC) and R.Renita (MIC) carried out the experiment, provide material and beneficial supports for the study; Riska (MIC), carried out the experiment, edited the manuscript provide material and beneficial supports for the study; ED.Husada (MAC) carried out the experiment, analyze the data the manuscript, write the manuscript.

Figure 1 .
Figure 1.The GC -MS chromatogram of total intensities of all mass spectral peaks detected in the leaves of Garcinia intermedia with air-dried (A), freeze (B), and oven (C) drying methods.

Figure 2 .
Figure 2. The GC -MS top peak of identified compounds in the leaves of Garcinia intermedia with air-dried (A), freeze-drying (B), and oven drying(C) methods.

Table 1 .
The list of identified metabolomic compounds in leaves of Garcinia intermedia.

Table 2 .
The summary of Friedelan-3-one in various species and its potential use.