Analysis of Volatile Components in Fresh Fruit of Morinda citrifolia L. from Different Origins Based on HS-SPME-GC/MS

Dan WANG, Qian JIANG, Qinglong WANG, Chao YUAN, Maoyuan WANG, Zuowang FAN, Xiaolu CHEN, Fulai YU

Abstract [Objectives] This study was conducted to clarify the differences of volatile components in fresh fruit of Morinda citrifolia L. from different origins.

[Methods]The method of HS-SPME-GC/MS detection was used to determine the volatile chemical components in fresh fruit of M. citrifolia.

[Results] 52， 52 and 45 volatile components were identified from the fresh fruit of M. citrifolia from Xisha， Wanning and Haikou， respectively. Among them， the number and content of the identified esters were relatively high， mainly methyl caprylate， ethyl caprylate， 4-pentenyl caproate， 3-methylbuten-2-enyl caprylate， methyl caproate， and ethyl caproate. There were also fatty acids， alcohols， phenols， ketones， aldehydes and other substances. There were 33 common ingredients in the fruit from the three origins， mainly including caprylic acid， caproic acid， capric acid， methyl caprylate， ethyl caprylate， 4-pentenyl caproate， 3-methylbutene-2-enyl caprylate， methyl caproate， ethyl caproate， methyl caprate and hexyl caproate.

[Conclusions]The types and contents of volatile components in fresh fruit of M. citrifolia from different origins were significantly different.

Key words Morinda citrifolia L.; Fresh fruit; Different origins; Volatile chemical components; GC-MS

Received： March 28， 2022  Accepted： May 29， 2022

Supported by Hainan Province Basic and Applied Basic Research Program （Natural Science） High-level Talents（No. 2019RC318）; Nanfeng Special Phase III： Investigation and Protection of Cultivated Land and Fishery Water Resources （NFZX2021）; the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences （No. 1630032022022）.

Dan WANG （1982-）， female， P. R. China， associate researcher， PhD， devoted to research and development of southern medicine resources.

*Corresponding author. E-mail： fulai.yu@163.com; chenxl@catas.cn.

Noni is the dry ripe fruit of Morinda citrifolia L.， also known as Haibaji， Luoli， Indian mulberry， which is native to the tropical islands of the South Pacific[1-2]. M. citrifolia is a perennial evergreen broad-leaved shrub or small tree that grows in tropical and subtropical regions. It has wild resources in Hainan， Taiwan and Xisha Islands in China， and there have been a large area of cultivation and introduction in Hainan and Xishuangbanna， Yunnan at present[3]. M. citrifolia fruit is rich in a variety of essential amino acids， vitamins， trace elements， polysaccharides and other nutrients and rich active substances， such as alkaloids， anthraquinones， polyphenols， fatty acids， lignans， polysaccharides， sterols， flavonoids and terpenoids， which have antibacterial， anti-tumor， antioxidant， anti-aging， anti-inflammatory， analgesic， blood pressure lowering， blood vessel protection， liver protection， alcoholic liver damage prevention and immune enhancement effects. As early as 2 000 years ago， M. citrifolia was used by the Polynesians to treat various diseases， and was hailed as "the holy fruit of the millennium"[4-10]. As a new health-care ingredient， M. citrifolia is increasingly popular in Western society. In May 2010， according to the Food Safety Law of the Peoples Republic of China and Administrative Measures for New Resource Foods， M. citrifolia fruit pulp was approved as a new resource food. M. citrifolia fruit has a special and strong smell. It is rich in nutrients and has good health care function and economic value. Therefore， the research on its chemical formation includes the development and utilization of M. citrifolia. At present， relevant researches on the nutrient composition， quality analysis and chemical composition analysis of M. citrifolia fruit have been reported[11-13]. Huang et al.[14] studied the quality standards of M. citrifolia medicinal materials produced in Hainan， including extracts， total ash， polysaccharides， etc.， but did not analyze their volatile components. Gong et al.[15] analyzed the volatile components of fresh M. citrifolia fruit， but the production areas of the fresh M. citrifolia fruit was unknown. Yang et al.[16] analyzed the chemical constituents of volatile oil in M. citrifolia fruit produced in Danzhou， Chengmai and Wanning， Hainan by the GC-MS method. In this study， we analyzed and identified the volatile components in M. citrifolia produced in Xisha， China and introduced and preserved in Danzhou， as well as in the fresh fruit of M. citrifolia from Haikou and Wanning， aiming to provide a reference for the evaluation of M. citrifolia fruit quality and data support for the selection of M. citrifolia planting areas.

Materials and Methods

Materials

The Haikou M. citrifolia samples were collected from Chinese Academy of Tropical Agricultural Sciences. The Wanning M. citrifolia samples were purchased from Wanning Lumeiying Biotechnology Co.， Ltd. The Xisha M. citrifolia samples were collected from Xisha， Hainan， and were introduced and preserved in Danzhou Nanyao Germplasm Resource Garden， Tropical Crop Germplasm Research Institute， Chinese Academy of Tropical Agricultural Sciences.

Experimental instruments

Agilent gas chromatography-mass spectrometer 7890B-5977B （Agilent， USA）; solid-phase microextraction device and extraction head （75 μm， CAR/PDMS， Supelco， USA）.

Methods

Analysis method of volatile component

The volatile chemical components in M. citrifolia fruit were determined by the HS-SPME-GC/MS detection method. First， a 1.00 g of fresh M. citrifolia fruit sample was accurately weighed and put into an SPME sample bottle. The injection port was aged at 250 ℃， and the extraction head at 45 ℃ was inserted into the sampling bottle for 20 min of adsorption. Next， GC/MS detection was carried out with a DB-WAX chromatographic column （30 m×250 μm×0.25 μm）. The temperature of the injection port was 250 ℃， and the heating program was started with an initial temperature at 40 ℃， which was held for 5 min and then increased at 3 ℃/min to 120 ℃， which was held for 3 min and then heated at 6 ℃/min to 230 ℃， which was held for 5 min.  The carrier gas （He） flowed at 1.00 ml/min under a pressure of 7.069 9 psi. The split ratio was 20∶1. The ion source used was EI source with a temperature at 230 ℃; the quadrupole temperature was 150 ℃; the electron energy was 70 eV; and full wavelength scanning was adopted within a scanning range of 30-400 m/z at a scanning speed of 781 u/s. The mass spectra of the separated chemical components were searched by computer， and analyzed by manual operation through searching of and comparison with standard spectra. The peak area normalization method was used for the identified chemical components to determine the relative content of each chemical component. Data analysis was performed using Pegasus software.

Results and Analysis

GC-MS total ion chromatograms of volatile components in fresh M. citrifolia fruit from different origins

Fig. 1 shows the GC-MS total ion chromatograms of volatile components in M. citrifolia fruit from Xisha， Wanning and Haikou.

Analysis of volatile components in fresh M. citrifolia fruit from different origins

It can be seen from Table 1 that 52， 52 and 45 volatile components were identified from the fresh M. citrifolia fruit from Xisha， Wanning and Haikou， respectively. Among them， the number of identified ester components was the largest， 33， 34 and 31 （see Table 2）， respectively， accounting for 63.46%， 65.39% and 68.89% of the total number of components， respectively， and the total content of ester components was also the largest， 60.33%， 65.81% and 68.02%， respectively. There were 33 common ingredients in the fruit from the three origins， mainly including fatty acids such as octanoic acid， caproic acid and capric acid， and esters， including methyl caprylate， ethyl caprylate， 4-pentenyl caproate， 3-methylbuten-2-enyl caprylate， methyl caproate， ethyl caproate， methyl decanoate and hexyl caproate.

The results of this study showed that the volatile components in fresh M. citrifolia fruit were mainly esters and fatty acid compounds， most of which have aromatic odors， are often used in food and spices， and have certain biological activity. It can be seen from Table 1 that caprylic acid， caproic acid and capric acid were fatty acids contained in M. citrifolia fruit from the three production areas， and the contents were relatively high. Among them， the caproic acid contents in M. citrifolia fruit from Xisha， Wanning and Haikou were 12.095%， 13.177% and 8.794%， respectively. Caproic acid is a fatty acid with six carbon atoms， and it is a colorless oily liquid at room temperature. Caproic acid is an edible spice that is allowed to be used in China. It is used to prepare various flavors and as a raw material for synthesizing other food spices. Moreover， caproic acid also has an inhibitory effect on Escherichia coli and Staphylococcus aureus， and can destroy the cell membrane structure of S. aureus[17]. Various esters derived from caproic acid are also commonly used in food additives， especially ethyl caproate， which has a pleasant smell and has the aroma of koji and pineapple， and is often used in food flavoring， in the preparation of tobacco flavors， and in flavorings for koji wine， as well as in the flavors of daily necessities such as cosmetics and spices[18]. In addition， the contents of methyl caproate in the fresh M. citrifolia fruit from the three origins were relatively high， at 7.147%， 10.088% and 6.676%， respectively.  Methyl caproate， with a pineapple-like aroma， is also an edible spice allowed in China. It is mainly used to prepare flavors such as pineapple and apricot essence. The caprylic acid contents in fresh M. citrifolia fruit from the three origins were 6.668%， 14.348% and 17.802%， respectively.   Caprylic acid is a straight-chain carboxylic acid with eight carbons. It is a colorless oily liquid at room temperature with a slightly uncomfortable odor. It can be used to manufacture dyes， medicines， spices， etc. As a practical spice we use， caprylic acid can also be used as a preservative， bactericide， foaming agent， defoaming agent， etc. In addition， caprylic acid also has anti-tumor effects[19]， inhibits Microcystis aeruginosa[20]， and can improve blood cholesterol concentration by reducing the absorption of exogenous cholesterol in the intestinal tract of mice， as well as promoting the excretion of total cholesterol in mouse feces[21]. Moreover， methyl caprylate and ethyl caprylate derived from caprylic acid are all contained in fresh M. citrifolia fruit from the three origins， and the contents were relatively high， but the contents of different origins varied greatly. The contents of methyl caprylate and ethyl caprylate in M. citrifolia from Xisha were 5.476% and 6.021%， respectively， 18.765% and 16.976% from Wanning， and 17.680% and 5.985% from Haikou. Methyl caprylate is an oily liquid with strong wine， fruit and citrus aromas. It is used in the preparation of edible flavors such as pineapple， berry and fruit flavors， and can also be used as an important raw material and intermediate for the synthesis of cosmetics and drugs[22]. Ethyl caprylate is a colorless and transparent liquid， occasionally with a slight yellow color， with a pineapple-like fragrance and sweetness. It can be used as a flavoring agent for orange， strawberry， pineapple and other flavors. Ethyl caprylate has the aroma of brandy and is mainly used in the manufacture of condiments and spices. Ethyl caprylate plays a very important role in the formation of liquor aroma and is prevalent in liquors of different flavor types[23]. Compared with caproic acid and caprylic acid， capric acid had a lower content. Capric acid is a ten-carbon fatty acid， which can be white crystal， or transparent and colorless or light yellow liquid. It is used in dyes， fragrances， greases， medicines， perfumes， professional soaps and synthetic rubbers. Capric acid has the functions of lowering blood sugar and regulating blood lipids. It may also help balance the bodys insulin levels to prevent insulin resistance[24-25]. It can be seen that the volatile oil of M. citrifolia fruit contains a variety of flavor substances with aroma， which is the reason for its special aroma， and these flavor substances often have a variety of biological activities， which is also the material basis of M. citrifolias health care effect.

Conclusions and Discussion

In this study， for the first time， the volatile components in M. citrifolia， which originated in Xisha， China and were introduced and preserved in Danzhou， and fresh M. citrifolia fruit from Haikou and Wanning， were analyzed and identified. The study found that there were significant differences in the types and contents of volatile components in fresh M. citrifolia fruit from the three origins. Among them， the types and contents of volatile components in M. citrifolia fruit from Xisha and Wanning were the most， followed by Haikou. There were 37 common components in the volatile components of fresh M. citrifolia fruit from Xisha and Wanning， 34 common components from Xisha and Haikou， and 43 common components from Wanning and Haikou.   Ester compounds were identified to have the highest quantities and contents from the three origins， among which 3-methylbuten-2-enyl caprylate， methyl caprylate and ethyl caprylate were higher in content. Fatty acids are another type of important compound in fresh M. citrifolia fruit， the main components are caprylic acid， caproic acid and capric acid. The types and contents of volatile components in fresh M. citrifolia fruit from the three origins were quite different， and the possible reasons might be related to the differences in the origin of M. citrifolia source， the harvest time， and the climate of the origin. The effects of chemical composition differences on the biological activity of M. citrifolia still needs further research.

Compared with previous research results， it is found that the types and contents of volatile components in M. citrifolia fruit are quite different. Gong et al.[15] determined by GC-MS that the volatile components in fresh M. citrifolia fruit were mainly fatty acids and esters， and there were 21 compounds in total， namely fatty acids， esters， alkanes， aldehydes and ketones， of which fatty acids were the main volatile components， accounting for 43.34%， including  bis（2-ethylhexyl）phthalic acid， octadecenoic acid， n-hexadecanoic acid and caprylic acid with higher contents. In addition to fatty acids and esters， this study also found that fresh M. citrifolia fruit contained alcohols， phenols and other compounds， and the main chemicals were esters with the highest content， while fatty acids were mainly caprylic acid， caproic acid and capric acid. Bis（2-ethylhexyl）phthalic acid， octadecenoic acid and n-hexadecanoic acid were not detected. The differences between the above results may be due to the differences in the preparation methods of the materials used in the two assays. In the above study， 95% ethanol was used for diafiltration extraction， followed by concentration， extraction with ether and evaporation to dryness， while this study directly used fresh M. citrifolia fruit without any treatment. Yang et al.[16] prepared the volatile oil of fresh M. citrifolia fruit from three origins by steam distillation. The study found that the fruit from the three origins of Danzhou， Chengmai and Wanning were determined by GC-MS to have 26， 20 and 19 chemical components， respectively. Fatty acids were the main chemicals， in which caprylic acid has the highest content， and esters， alcohols and phenolic compounds were also contained. The common ingredients of the three origins were mainly caprylic acid， capric acid， caproic acid， methyl caprylate， stearic acid and palmitic acid. This study also found that 52， 52 and 45 volatile components were identified from the fresh M. citrifolia fruits from Xisha， Wanning and Haikou， respectively. The main chemical substances were esters with the highest content， while the fatty acids were mainly caprylic acid， caproic acid and capric acid. Stearic acid and palmitic acid were not detected. The differences between the above results may be due to the different preparation methods of the materials used in the two determinations. The above study used steam distillation to extract volatile oil from fresh M. citrifolia fruit， while this study directly used fresh M. citrifolia fruit without any treatment. Huang et al.[26] used GC-MS and GC to qualitatively and quantitatively analyze the fatty acids in M. citrifolia after methyl esterification of the ether extract of M. citrifolia fruit. A total of 17 fatty acids were isolated， among which the saturated fatty acids were mainly hexadecanoic acid （palmitic acid）， octadecanoic acid （stearic acid）， caprylic acid， caproic acid， butyric acid， capric acid， heptadecanoic acid and eicosanoic acid. The unsaturated fatty acids were mainly linoleic acid， oleic acid， elaidic acid and palmitoleic acid. There was a great difference from the results of this study， and no unsaturated fatty acids such as linoleic acid， oleic acid and palmitoleic acid were found in the measurement results of this study. The reason for the large difference should be mainly due to the ether extraction and methyl esterification carried out in the above study， resulting in the differences between the identified compounds and those of the present study.

M. citrifolia has become one of the best-selling health products in the world due to its unique functional properties. Hainan Island is one of the most suitable areas for the introduction and cultivation of M. citrifolia. Studying the volatile components of M. citrifolia fruit from different origins in Hainan Island is helpful to the analysis of its product quality， and can provide a reference for the development and utilization of M. citrifolia， as well as for the selection of M. citrifolia planting sites.

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