Improved Method of Recrystallization of Zingiber zerumbet Extracts to Obtain Zerumbones

1. Introduction

Plant foods, spices and herbs have ruled for generations as traditional medicines, ayurvedic, perfumes, preservatives, aroma enhancers for food and beverages, seasonings and flavourings and will continue to partake in our daily lives as they contain a barrage of phytochemicals (Shahrajabian et al., 2019). In this era, an increasing number of people are using herbs and spices as home remedies to alleviate simple ailments and to promote one’s health. In addition, with burgeoning metabolic syndromes, complementary and alternative medicines are being sought after to manage such diseases and to prevent worsening of these syndromes. Furthermore, there are a number of publications in the pharmaceutical industry that have considered these phytochemicals beneficial to human health.

Zingiberaceae is the largest monocotyledonous family of the plant kingdom found in tropical and subtropical regions of the world. The members of this family, such as Zingiber officinale (ginger), Curcuma longa (turmeric), Zingiber zerumbet (bitter ginger) and Elettaria cardamomum (cardamom), are used in complementary medicine, agriculture, in cuisines and for ornamental purposes (Zakaria et al., 2011). The genus Zingiber contains approximately 85 species. This medicinal plant is cultivated extensively because of its low planting costs. Besides its wide usage as a spice, ginger which can be taken raw or cooked has been used traditionally as treatment for indigestion, flatulence, gastritis, gastro-esophageal reflux, dyspepsia, nausea, morning sickness, hangover, common cold, sinusitis, sore throat, fever, arthritis, rheumatoid arthritis, colic, stomach cramps, toothache, tension headache, migraine headache, asthma and many more (Ghasemzadeh et al., 2017; Singh et al., 2014; Li et al., 2020; Silva et al., 2018; Noor and Sirat, 2016; Rahman et al., 2014). Even products these days have added brown sugar with ginger for a more sweetness to spiciness taste and with that, making it more palatable to consume.

Zingiber zerumbet (L.) Smith, one of the most cultivated Zingiberaceae family which is native to SouthEast Asia, especially India, Indonesia and Malaysia, has made its way into our lives for generations as a spice and most widely used condiments in various cuisines and beverages throughout Asia (Ghasemzadeh et al., 2016; Rahman et al., 2014; Shahrajabian et al., 2019). Due to its many types of phytochemicals found in Zingiber zerumbet, it is regarded as one of the spices in preventing or managing certain medical conditions (Ghasemzadeh et al., 2017). It is also well spread throughout tropical and subtropical regions of other countries such as Sri Lanka, Nepal, China, Japan and other parts of the world (Rahman et al., 2013). Zingiber zerumbet is a warm-season perennial, tuberous root plant adapted for growth in damp and shaded parts of lowlands or hill slopes (Khalid et al., 2011; Shahrajabian et al., 2019). It is observed that they tend to have the best growth spurts at temperatures of 25-28 °C (Tan et al., 2018).

There are various names being used in different countries with regards to Zingiber zerumbet such as bitter ginger, wild ginger, shampoo ginger, bitter ginger, awapuhi, lempoyang as it is commonly known as in Malaysia and Indonesia; parsu kedar, ghatian, and yaiimu in India, hong qui jiang in China and the list goes (Sachin et al., 2017; Shahrajabian et al., 2019; Singh et al., 2019). All the plant parts such as the flower, leaves and rhizomes respectively carry multiple health promoting effects and have been researched by many as they contain medicinal benefits (Padalia et al., 2018; Sachin et al., 2017). Briefly, the fragrant leaves are used to enhance the taste, colour and odour of cuisines and the red pine cones which is the flower is used for softening and bringing shininess to the hair (Jalil et al., 2015). Rhizomes with a light brown skin have a fibrous and scaly root and a pungent scent (Singh et al., 2014). Powdered rhizome is used to treat ear infections and toothache as the stinging nature of ginger tends to curb pain (Ghosh et al., 2011). Rhizomes are the ones that play a part in food flavouring in cuisines and traditional medicine amongst the people across the globe (Nik et al., 2009; Singh et al., 2019).

The oil of the rhizome has attracted the most attention from researchers for its potential human health benefits as it is a complex mixture of terpenoids, comprising the largest subgroup sesquiterpenoids; mainly zerumbone the most active constituent followed by humulene, humulene oxides, βcaryophyllene and α-caryophyllene and varying proportions of monoterpenoids; camphene, sabinene, myrcene, amongst others (Akhtar et al., 2019; Azelan et al., 2018; Kiyama, 2021; Koga et al., 2016; Singh et al., 2019; Zhang et al., 2018). Recrystallization of the oil of Zingiber zerumbet with an inorganic solvent is an effective way to produce the pure active compound, zerumbone. Zerumbone has three double bonds, two conjugated and one isolated, as well as a double conjugated carbonyl group in the 11-membrane ring structure (Kitayama et al., 2001). The boiling and melting point of zerumbone are 321–322◦C at 760 mm Hg and 65.3◦C, respectively (Kitayama et al., 1999). Zerumbone (2E,6E,10E)-2,6,9,9- tetramethyl cycloundeca-2,6,10-trien-1-one), a monocyclic sesquiterpene ketone that has reported to act as an anti-inflammatory, antimicrobial, anticancer, antioxidant, antipyretic, analgesic, cyclooxygenase-2 suppressant properties, antibacterial, anti-allergic, anti-ulcer, antioxidant, antinociceptive, antiplatelet aggregation, and hepatoprotective (Foong et al., 2018; K. Kalantari et al., 2017; Md et al., 2018; Padalia et al., 2018; Sulaiman et al., 2009)

According to Khalid et al. (2011), upon testing zerumbone on models of nociception, found that the extract of Zingiber zerumbet acquires central and peripheral antinociceptive activity. It is also further reported by Zakaria et al. (2010), that the involvement of zerumbone in inhibition of prostaglandin, histamine and opioid-mediated among others manages to exhibit anti-inflammatory and antinociceptive activities. Furthermore, Chia et al. (2020), suggested that due to the mode of action that zerumbone plays in the antiinflammatory receptors pathway, zerumbone is beneficial in managing neuropathic pain in combination with conventional therapy. However more studies are needed to improve the current approaches in the treatment of neuropathic pain. As demonstrated by Somchit et al. (2012), the extract of Zingiber zerumbet showed lower anti-inflammatory activity as compared to the pure active compound, zerumbone. This also goes to show that zerumbone has a potent analgesic effect which is comparable to that of a non-steroidal anti-inflammatory drug (NSAID). In vitro and in vivo studies by Chien et al. (2016) and Tian et al. (2020) have concluded that zerumbone extracted from Zingiber zerumbet exhibited an anti-inflammatory effect on induced arthritis in mice as it involves the downregulation of overly-secreted prostaglandins which are responsible for inflammation. In addition to the benefits of the pure active compound of Zingiber zerumbet, various preclinical studies have indicated that zerumbone managed to repress the IL-6/JAK2/STAT3, PI3K/AKT/mTOR pathways and downregulate the expression CXCR4, activation of NF-κB, and other oncogenic proteins (Girisa et al., 2019). Various cancers such as brain, breast, colon, liver, and lung can be managed as cancer cell proliferation will be inhibited due to induction of cell cycle arrest and apoptosis (Girisa et al., 2019). However, Jalili-Nik et al. (2020) also lamented that more preclinical trials need to be undergone for a better understanding of the anti-cancer properties of zerumbone.

Since the extracts of Zingiber zerumbet possess many pharmaceutical benefits, it is only necessary to delve in its benefits by optimizing the extraction of its rhizomes. Due to the presence of various mixtures of terpenoids in the oil of rhizomes as mentioned previously, it is of utmost importance to recrystallize to obtain the pure active compound of zerumbone crystals.

2. Materials and Methods

2.1. Materials

Crude material, fresh rhizomes of Zingiber zerumbet, were procured from a local market in Chow Kit, Malaysia. Fresh rhizomes were identified by the Dr. Mohd Firdaus Ismail, Institute of Bioscience, Universiti Putra Malaysia (UPM), Serdang, Malaysia (MFI 0180/20). Analytical grade hexane was used for decanting of extraction compound. Distilled water from filtration system in Physiology Lab, Universiti Putra Malaysia (UPM), Serdang was used with the solvent to prepare the extraction compound. HPLC was done at Natural Products Division, Forest Research Institute Malaysia (FRIM), Selangor. All other equipment used was from Physiology Lab, Universiti Putra Malaysia (UPM).

2.2. Preparation of Extraction

Firstly, 6 kg rhizomes of Zingiber zerumbet were cleaned and cut into pieces approximately 1 cm lengths. The cut rhizomes were then oven-dried for three days at 50°C and weighed thereafter. Two batches of oven-dried cut rhizomes, each weighing 1 kg were prepared. The first batch of 1 kg of oven-dried cut rhizomes were ground into powder by using a commercial grade grinder (Model EBM-9182, ELBA Malaysia) and weighed again after being ground. And the second batch of 1 kg of oven-dried cut rhizomes were extracted as intact.

2.3. Extraction of Zingiber zerumbet

Both forms of dried rhizomes were extracted by hydrodistillation method using a Clevenger-type apparatus (Noor et al., 2016). Briefly, the crude materials were added into a round bottom flask seated in a heating mantle. Then, 3L of distilled water was added into the flask, to immerse the crude materials. The round bottom flask was connected to the distiller and finally to the condenser. The pipes were connected to the running filtered tap water and the cooler was set at 9°C. The contents were boiled at 100°C. Hydrodistillation was stopped when no pale yellowish oil crude extracts were seen at the collecting tube. Hydrodistillation was conducted for 6 hours for both forms of crude plant materials respectively. Pale yellowish crude extracts, a mixture of oil and water that were collected from both forms of dried materials were stored in conical tubes at 4°C respectively.

2.4. Optimization of Recrystallization and Isolation of Zerumbone

After hydrodistillation, using a glass separatory funnel with stopcock, a liquid-liquid extraction using hexane as a solvent was performed for both pale yellowish crude extracts that were obtained from both forms of crude plant material (Al-Amin et al., 2019). Briefly, crude extracts were washed with distilled water twice, each with 10 ml. Then, 10 ml of hexane was added into the separatory funnel and secured with a stopper. The funnel was shaken a few times to release the pressure that had formed in the funnel and it was allowed to stand for a few seconds for the separation to be settled. The aqueous phase was drained into a flask. In a separate beaker, the remaining liquid was collected. The funnel was flushed with an additional 5 ml of hexane and the step was repeated twice. To evaporate the hexane, the liquid was boiled to isolate the oil. It is noticed that the liquid boiled at 67.5°C. The liquid was poured in another beaker and the previous beaker was washed with a further 5 ml of hexane twice. Heating process repeated. The liquid was again poured in another beaker and the previous beaker was washed with a further 5 ml of hexane twice and heating process continued thereafter. On low heat, when the thermometer that was placed in the beaker showed 90°C and no evaporation activity was seen, the heating process and evaporation of hexane was stopped. After decanting, the extracts from both forms were left in the fume hood for a day, to dry and to spontaneously crystallize respectively.

After which, pure zerumbone crystals was further dried by vacuum filtration. Using a Buchner funnel, zerumbone crystals were placed on a wet filter paper which was cut to fit into the funnel. The end of Buchner funnel was then placed into the rubber funnel followed by snugging it tightly onto the mouth of the filtration flask. The vacuum hose was connected to the filtration flask and the vacuum was switched on for 30 minutes to remove any moisture from the crystals. Pure zerumbone crystals from respective forms of dried Zingiber zerumbet were weighed in vials, sealed in aluminium foil and kept at 4°C, respectively. The percentage of yield of pure active compound, zerumbone was calculated based on weight to weight (^w/w) as shown in Equation 1

$$Yield\left(\%\right)={\displaystyle \frac{W_{PE}\left(g\right)}{W_T\left(g\right)}}\times 100$$

(1)

Where W_PE is the weight of pure extract after recrystallization and W_T is the weight of dried/ and ground Zingiber zerumbet.

2.5. Identification of Zerumbone

Preparation of samples were done by adding 2 ml of methanol to dissolve 1 mg of sample in a vial. The mixtures were then placed in an ultrasonic machine for 15 minutes. The resulting solutions were filtered using a PTFE 0.45 μm cartridge prior to analysis. Samples were then analyzed to ascertain the purity of Zerumbone with the high-performance liquid chromatography (HPLC) system (Waters 2535 quaternary gradient using a PTFE 0.45 μm cartridge prior to analysis. Samples were then analyzed to ascertain the purity of zerumbone with the High-Performance Liquid Chromatography (HPLC) system (Waters 2535 quaternary gradient pump, Waters 2707 autosampler and Waters 2998 PDA). A HPLC Phenomenex Luna PFP (2) (5 μm, 250 mm x 4.6 mm) was used and for elution, an isocratic solvent consisting of 2 types of solvent which were 25% A (0.1% aqueous formic acid) and 75% B (acetonitrile) were used (Foong et al., 2018). The flow rate used was 1.0 ml/min and the injection volume was 10 μl. The samples were analyzed by ultraviolet detection at a wavelength of 280 nm (Rahman et al., 2013). The retention times and UV spectra of the major peaks were determined and compared with the standard zerumbone.

3. Statistical Analysis

The data represents three independent experiments, and the results were expressed in mean ± SD. The differences between two groups were analyzed using independent t-test. Graph pad version 8.0.2 was used to perform all statistical data. The significance level was set at p<0.05.

4. Results

4.1. Extraction of Zingiber zerumbet

After being dried in the oven for three days, the weight of 6 kg of freshly cut rhizomes was reduced to 3.03 kg. Only 1 kg of dried cut rhizomes was taken to prepare two batches, one ground and the other left intact. The weight of powdered dried Zingiber Zerumbet was reduced to 900 g from the initial 1 kg, after grinding and had to be made up to 1 kg separately to compare the yield with intact Zingiber zerumbet which was 1 kg. Figure 1(a) shows the plant material, Zingiber zerumbet and (b) freshly cut Zingiber zerumbet.

After 6 hours of hydrodistillation, pale yellowish crude extracts that were collected from dried intact rhizomes and ground dried rhizomes of Zingiber zerumbet were 57.50 g/kg and 30.0 g/kg respectively. Significant difference (p<0.05) by independent t-test was exhibited in terms of yellowish crude extracts collected between ground dried rhizomes and dried intact rhizomes after hydrodistillation. Figure 2(a) depicts the apparatus used for hydrodistillation and the example of (b) pale yellowish crude extracts obtained after extraction. Crude extracts from both powdered dried and intact dried Zingiber Zerumbet had a mixture of oil and water.

In order to remove the water from the crude extracts, separatory funnel was used to drain the oil and water in a separate beaker. Distilled water was also added to completely wash the extracts and to make sure only the oil was collected. Upon repeated decanting with hexane, a method also suggested by Noor and Sirat, (2016) and Rahman et al. (2013), a dark yellowish oil which had a warm aromatic scent was produced and both the forms crystallized rapidly. After vacuum filtration to completely remove any moisture from the crystals, off-white zerumbone crystals of 9.87 g/kg rhizomes (yield,0.99%^w/w) and 6.97 g/kg (yield, 0.70%^w/w) rhizomes was obtained from dried intact rhizomes and ground dried rhizomes respectively. Independent t-test showed significant difference (p<0.05) of weight of zerumbone crystals from dried intact rhizomes and dried grround rhizomes. Figure 3 depicts the pure zerumbone crystals obtained after vacuum filtration.

The purity of zerumbone determined by the HPLC analysis for ground and intact samples were 98.51% and 99.68% respectively demonstrating a major peak with retention time of 6.126 minutes from ground sample and 6.133 minutes from intact sample with an insignificant difference. Figure 4 and Figure 5 illustrates the respective HPLC analysis of zerumbone.

5. Discussion

The most used extraction method of plant materials has been hydrodistillation as it isolates non-water-soluble compounds with high boiling points (Akhtar et al., 2019; Huong et al., 2019; Katayoon Kalantari et al., 2017). The powdered dried rhizomes produced a lesser yield of yellowish crude extracts as compared to the dried intact rhizomes. That is because during the process of grinding, only 1% of the energy transmitted to the grinder is used for size reduction whilst 99% of energy is converted into heat (Jung et al., 2018; Murthy et al., 1999; Rossi et al., 2020). The elevated temperature causes not only a significant loss of its volatile oil but also intensified the colour of the compound. Moreover, with every size reduction, more surface area is being increased and more cells are being damaged resulting in the availability of the oil to be at the surface (Liu et al., 2018; Murthy et al., 1999). It was also observed that the powdered dried rhizomes were easier to extract, and it produced oil extracts faster than the dried intact rhizomes. This is due to the oil pockets in dried rhizomes breaking down during grinding which makes oil from powdered rhizomes to be easily extracted (Saxena et al., 2018). After grinding the 1 kg of dried cut rhizomes, the weight was reduced to 900 g and the powdered rhizomes appeared warm to touch and exuded a strong smell. The process of grinding creates high temperatures which in turn melts the fat or oil present in the compound resulting in it sticking to the surface of the grinder (Saxena et al., 2014; Sharma et al., 2016). This further explains the loss of weight of the compound after grinding. Therefore, these reasons conclude a lesser yield of powdered dried rhizomes as compared to dried intact rhizomes.

Based on the GC-MS conducted by Tian et al. (2020) as depicted in Figure 6, there are thirty-six compounds identified representing 98.8% of essential oil from dried Zingiber zerumbet consists mainly zerumbone (41.9%), followed by α-humulene (29.4%), humulene oxide I (6.0%), humulene oxide II (3.9%), camphene (3.9%), β-caryophyllene (2.5%), camphor (2.4%), caryophyllene oxide (2.1%), and 1,8-cineole (1.6%). And as mentioned previously, only zerumbone that exerts pharmaceutical benefits is needed. Therefore, recrystallization with hexane is an effective method to obtain pure zerumbone off-white crystals (Zhang et al., 2018). The apparatuses were flushed with hexane at least three times to remove any traces of crude oil extracts to maximize yield of crystals. As hexane easily evaporates into air and has a boiling point of 69°C, heating of the oil extracts was continued to 90°C until no evaporation was seen to ensure only zerumbone was acquired (Karthikeyan et al., 2019; Perez-Hurtado et al., 2017).

Even though, previous authors did not disclose the method of recrystallization in depth but in this study, the curated recrystallization steps in this study have indeed produced more yield of zerumbone crystals as compared to 1.30 g/kg as reported by Albaayit et al. (2020) and Rahman et al. (2013) respectively. Akhtar et al. (2019) produced 87.40 mg of zerumbone crystals from 1.75 kg of Zingiber zerumbet rhizomes. On the contrary, the closest yield to our study is the one reported by Al-Amin et al. (2019) who produced 9.10 g of zerumbone from 1.30 kg of dried rhizomes, through vacuum liquid chromatography using 5 types of solvent through 5 fractions. However, in our study, the use of only one solvent and its simple method of recrystallization renders the proposed method to be cost effective and applicable for large scale. As also discussed previously, due to the high temperature that emanates during grinding, plant compounds are damaged resulting in low volatile oil which in turn produces a lesser yield of active compound crystals (Cirri et al., 2012; Liu et al., 2018; Saxena et al., 2014). Therefore, oil from dried intact rhizomes produces more white crystals of zerumbone as opposed to the oil from ground dried rhizomes.

6. Conclusions

This study concluded that ground dried Zingiber zerumbet produced significantly lesser yield of zerumbone compared to the intact dried Zingiber zerumbet. Most importantly, the optimization of recrystallization of essential oil which is an effective method in acquiring significantly more yield of pure active compound of zerumbone crystals also showed satisfactory purity of 99.68% and 98.51% and from intact and ground and dried Zingiber zerumbet respectively. The zerumbone crystals obtained through the recrystallization method employed in this study were utilized throughout this research.