Supercritical Fluid Extraction (SFE) of Malaysian Wild Ginger Zingiber puberulum Inflorescence

Introduction

Malaysian natural product plants have been studied extensively. These include medicinal plant species from Andrographis^1,2,3,4, Musa⁵, Plumeria^6,7, Citrus^8,9, Cymbopogon¹⁰, Garcinia^11,12, Artocarpus¹³, Kaempferia¹⁴   and Alpinia¹⁵.  Besides medicinal plants, fish products particularly ω-fatty acids have been studied extensively^16,17. However, plant species of  Zingiberaceae continue to attract much phytochemical interests due to their culinary uses, besides their biological and pharmaceutical activities. Recently, researcher from Universiti Putra Malaysia (UPM) has found a natural substance from Z. zerumbet  to treat and prevent cervical cancer¹⁸. With the discovery of the new medicine, the cost of cancer treatment would be lower than the cost of using imported drugs. Unfortunately, of the Zingiberaceae family, Z. puberulum  is less known and studied compared to the common ginger, Z. officinale.  Only work by Sirat and co-workers¹⁹ found that the extract of  Z. puberulum contained the labdane diterpenic constituents. Meanwhile, Theilade²⁰ documented the existence of Z. puberulum in Thailand ginger.

The supercritical fluid extraction (SFE) is a technology of interest to the food, cosmetics and pharmaceuticals industries, as an alternative to conventional methods, i.e. solvent extraction and  solid-phase microextraction (SPME)²¹. SFE employs the use of supercritical fluid as the extracting solvent replacing the use of conventional organic solvents. Many researchers have employed SFE to extract essential oils and oleoresins from ginger species. Nik Norulaini et al²² extracted zerumbone  from Z. zerumbet using SFE-CO₂ while Chen et al²³  have used Taguchi method in extracting  Z. officinale extract with SFE-CO₂. Based on these approaches, the present study aims to extract bioactive constituents from Z. puberulum using the SFE technique.

Experimental

Materials

Samples  of  Z. puberulum inflorescences were taken from Lojing, near Cameron Highland, Pahang, Malaysia and stored at – 4°C in the freezer for preservation purposes. The samples were washed, and chopped horizontally into smaller pieces. Samples used for SFE were  dried using a vacuum oven at 40°C for 48 hours to remove the water content and then ground into powder using a blender.

Extraction Of Volatile Components.

Supercritical Fluid Extraction (SFE)

Supercritical CO₂ was carried out using SFT-150 Supercritical Fluid Extraction/Reaction System (Supercritical Fluid Technologies Inc. Newark, Delaware, USA) comprising of a carbon dioxide cylinder, stainless steel vessel, 100 ml hand-tight sample vessel, air regulator, PID temperature controller and variable restrictor valve (back pressure regulator).

Approximately 10g of blended Z. puberulum  sample was placed in the sample vessel. The sample vessel was placed in the stainless steel vessel and allowed to equilibrate to the preset extraction temperature of 40°C. The high pressure pump compressed the CO₂ to the desired 48 Mpa pressure. The sample was subjected to 30 minutes static extraction (by closing the restrictor valve) followed by every 30 minutes dynamic extraction (by opening the restrictor valve). The essential oil collected in a pre weighted container was weighted after 3 hours of extraction.

Analysis  Of Volatile Components

Gas Chromatography-Mass Spectrometry Analysis (GC-MS)

1 µl of extract in dichloromethane was analyzed using PerkinElmer AutoSystem XL Gas Chromatograph (PerkinElmer, Shelton, CT, USA). The temperature was initially kept at 40°C for 1 minute,  then programmed  at 4°C per minute until it reaches 250°C, and then kept at the final temperature  for 5 minute. Helium gas was used as a carrier gas with a flow rate of 1 ml/min.   For identification of the chemical constituents in the samples, the PerkinElmer TurboMass Gold Mass Spectrometer was used. The compounds are identified by comparing the MS spectrum obtained with the standard library (NIST 2000)

Results and Discussion

Extraction

The supercritical fluid extraction (SFE) of the inflorescences of  Z. puberulum  gave dark yellowish viscous oil in 1.2% yield (w/w). Previous report found that by using simultaneous distillation extraction (SDE), a colourless non-viscous oil of Z. puberulum in 0.15% yield was produced²⁴. Similarly, the result showed that the higher yield and the more concentrated essential oils were extracted using SFE than SDE.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

Table 1 shows the volatile constituents identified in Z. puberulum extract by GC-MS technique.  It was found that the main constituents of  Z. puberulum extracted using SFE were palmitic acid (C16:0)  (65.7%), followed by 1-heptatriacotanol (16.9%) and oleic acid (C18:1) (15.%). This result verified that in this study SFE extracted the non volatile fatty acids along with the volatile compounds. According to Diaz-Morato et al.²⁵, using high pressure CO₂ densities may allow terpenes and oxygenated terpenes to be completely miscible in supercritical CO₂, but other non volatile compounds such as fatty acids, waxes and paraffins can also appear in the extract. Given that high pressure was used in SFE (48 Mpa), this explains why SFE extracts of  Z.  puberulum had high amount of fatty acids in it.  It was also proved  that high pressure is not recommended for complex matrices owing to the higher solubility of solutes when the pressure is elevated, resulting in complex extracts and difficult analysis as is in this case²⁶.

Table 1:  Volatiles identified in the essential oils of Zingiber puberulum  extracted using SFE

Peak	Compound	% relative
1	β-pinene	0.001
2	Undecanal	0.01
3	α-Terpineol	0.02
4	Decanal	0.01
5	cis-Geraniol	0.03
6	Isopinocarveol	0.01
7	Caryophyllene	0.07
8	α- Caryophyllene	0.05
9	Phellandral	0.03
10	β-Bisabolene	0.01
11	β -Farnesene	0.02
12	β-Sesquiphellandrene	0.03
13	Caryophyllene oxide	0.04
14	α-Farnesene	0.01
15	β-Elemene	0.14
16	α-Bisabolol	0.57
17	Tetradecanal	0.07
18	Palmitic acid	65.7
19	Phytol	0.43
20	Oleic acid	15.9
21	1-Heptatriacotanol	16.9

In this study, many of the volatiles identified in Z. puberulum oil  were similar to the volatiles in  Z. spectabile cultivated in Amazon²⁷.  However, the percentages were quite different: β-phellandrene, α-pinene and β-pinene were not the major components of the Zingiber spectabile  oil extracted by SFE, and neither were dodecanal, tetradecanol and α-pinene in SFE extracted oils of Zingiber puberulum. The reason could be attributed to difference in cultivation, geographical location and method of extraction.²⁸

Comparison of the compounds in the essential oils of Zingiber spectabile and Zingiber puberulum showed the presence of 7 similar compounds; undecanal, α-terpineol, phellandral, zingiberene, farnesene, caryophyllene oxide, 1-heptatriacotanol and phytol. This showed the connection between the different species in the same Zingiberaceae family.

Many of the bioactive compounds identified in the ginger flower had been known to have certain properties. β-sesquiphellandrene and ar-curcumene are the prime contributors to the characteristic ginger attribute while α-terpineol, neral, and geranial contribute to the lemony aroma of ginger oil, and may therefore be desirable additives to whole ginger oil to intensify its lemony or citrus character.   β-elemene, a sesquiterpene hydrocarbon found in abundance in Z. puberulum, is a novel anticancer drug, which has also been found in the Zingiber officinalis species²⁹. Thus, the results of the present investigation clearly indicate that the flower of wild ginger species possess important bioactivities which could be manipulated commercially.

Acknowledgement

The authors wish to thank International Islamic University Malaysia (IIUM) for providing the research grant (RMGS) to carry out this research. Thanks also to the staff of the Kulliyyah of Science (IIUM) for their technical assistance.

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