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Food Chemistry 132 (2012) 1150–1155
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Adaptogenic-active components from Kaempferia parviflora rhizomes Patcharee Pripdeevech a, Kitsada Pitija b, Chaiyong Rujjanawate c, Sermsakul Pojanagaroon d, Prasat Kittakoop e, Sugunya Wongpornchai b,⇑ a
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Center of Excellence for Innovation in Chemistry, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand c School of Health Science, Mae Fah Luang University, Chiang Rai 57100, Thailand d Loei Plant and Plant Production Material Technical Service Center, Phurua, Loei 42160, Thailand e Chulabhorn Research Institute, Chulabhorn Graduate Institute, Chemical Biology Program, Vibhavadi-Rangsit Road, Bangkok 10210, Thailand b
a r t i c l e
i n f o
Article history: Received 13 May 2011 Received in revised form 10 August 2011 Accepted 7 November 2011 Available online 22 November 2011 Keywords: Kaempferia parviflora Adaptogenic activity Adaptogenic herbs Terpenes GC–MS NMR
a b s t r a c t Kaempferia parviflora rhizome extracts obtained by maceration with hexane, chloroform, methanol, and ethanol were screened for their adaptogenic activities using swimming tests of mice. The effective adaptogenic extract dose was 500 mg/kg of body weight and was given orally once a day. Crude hexane extract showed significantly shorter mouse immobilisation time than those of the other and control extracts. This crude hexane extract was separated into three fractions by column chromatography. Among these fractions, the fraction rich in terpenoids possessed the highest adaptogenic activity and was comparable to that of the crude ginseng root powder used as a reference control. Therefore, terpenes contained in this fraction could be attributed to the decrease in exhaustion during the swimming of mice. There was no effect on body weight, heart, liver, kidneys, and adrenal glands of the mice. Chemical characterisation of this adaptogenic-active fraction by NMR and GC–MS showed germacene D, b-elemene, acopaene, and E-caryophyllene as major constituents. Accordingly, these terpenes are considered the adaptogenic agents of K. parviflora rhizomes. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Kaempferia parviflora Wall ex Baker is locally known as ‘‘Kra Chai Dam’’ in Thailand. It belongs to the Zingiberaceae family and is referred to as Thai ginseng (Sudwan, Saenphet, Saenphet, & Suwansirikul, 2006). Its rhizomes have been used for health promotion: as an anti-flatulent, for stomach discomfort and leucorrhea, as a diuretic and anti-dysenteric, and for the treatment of oral diseases (Chomchalow, Bansiddh, & MacBaine, 2003; Sudwan et al., 2006). The rhizomes have also been frequently used for treating gout, abscesses, colic disorder, and peptic and duodenal ulcers. A tonic drink made from K. parviflora rhizomes is commercially available and is believed to decrease impotent symptoms (Yenjai, Prasanphen, Daodee, Wongpanich, & Kittakoop, 2004). Additionally, rhizomes of K. parviflora have been used for treatment of allergy, gastrointestinal infection, fungal infection, and impotence (Pengcharoen, 2002). Due to the pharmaceutical properties of K. parviflora rhizomes, much attention had been paid to the study of the chemical composition of the rhizomes. Jaipetch, Reutrakul, Tuntiwachwuttikul,
⇑ Corresponding author. Tel.: +66 53 943341 5x120; fax: +66 53 892277. E-mail addresses: (S. Wongpornchai).
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0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.11.025
and Santisuk (1983) identified 10 constituents in the hexane extract of resin, or non-volatile oil, of K. parviflora rhizomes. These constituents are 5-hydroxy-7-methoxyflavone, 5,7-dimethoxyflavone, 5-hydroxy-7,40 -dimethoxyflavone, 5,7-dimethoxyflavone, 5,7 ,40 -trimethoxyflavone, 5,7,30 40 -tetradimethoxyflavone, 5-hydroxy3,7-dimethoxyflavone, 5-hydroxy–3,7,40 -tetramethoxyflavone, 5,3,7-trimethoxyflavone, and 5-hydroxy-3,7,30 ,40 -trimethoxyflavone. Herunsalee, Pancharone, and Tuntiwachwuttikul (1987) found 3,5,7,30 ,40 -pentamethoxyflavone, 5,3,7,40 -tetramethoxyflavone, 5hydroxy-7,40 -dimethoxyflavone, 20 -hydroxy-40 ,60 -dimethoxychalcone, and 20 -hydroxy-4,40 ,60 -trimethoxychalcone as the principle components in the crude chloroform extract of K. parviflora rhizomes. Trakoontivakorn et al. (2001) reported ()-hydroxypanduratin A, ()-panduratin A, sakuranetin, pinostrobin, pinocembin, and dihydro-5,6-dehydrokawain as the main constituents in the dichloromethane extract of K. parviflora rhizomes. Also, Sutthanut, Sripanidleulchai, Yenjai, and Jay (2007) found that 5,7-dimethoxyflavone and 5,7,40 -trimethoxyflavone were the major components of K. parviflora rhizomes, their contents being 21.68 and 9.88 mg per gramme of sample, respectively. Tewtrakul, Subhadhirasakul, Karalai, Ponglimanont, and Cheenpracha (2009) reported some active flavonoids isolated from K. parviflora rhizomes that possessed inhibitory activities against nitric oxide production which were 5-hydroxy-3,7-dimethoxyflavone, 5-hydroxy-7-methoxyflavone,
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5-hydroxy-3,7,40 -trimethoxyflavone, 5-hydroxy-7,40 -dimethoxyflavone, 5-hydroxy-3,7,30 ,40 -tetramethoxyflavone, 3,5,7-trimethoxyf lavone, and 3,5,7,40 -tetramethoxyflavone. Recently, antimutagenic and a-glucosidase inhibitory effects of constituents from K. parviflora have been reported by Azuma et al. (2011). The potent antimutagenic activity was demonstrated in relation to the presence of some major flavonoid constituents including 7-methoxyflavones, 5,7-dimethoxyflavone, 5,30 -dihydroxy-3,7,4-trimethoxyflavone, 3, 5,7-trimethoxyflavone, and 5-hydroxy-7-methoxyflavone. Adaptogens are the plants or any substances that appear to induce a state of non-specific increase of resistance of an organism to aversive stimuli that threaten to perturb internal homoeostases (Brekhman & Dardymov, 1969; Lazarev, 1947). These adaptogens increase tolerance to change in environmental conditions and resistance to noxious stimuli, such as exposure to cold, heat, pain, general stress, and infectious organisms. They have been claimed to arrest the ageing process and age-induced deterioration in physical and mental performance (Kannur, Hukkeri, & Akki, 2006). Examples of common adaptogenic herbs are dang shen (Codonopsis pilosula), licorice (Glycyrrhiza glabra), holy basil (Ocimum sanctum), ginseng (Panax ginseng), and noni (Morinda citrifolia). Their important medicinal properties have made these herbs distinct from other plant substances. The adaptogenic effect of K. parviflora rhizomes was investigated in this study using a rodent forced swimming capacity test. In order to explore the marker compounds responsible for this activity, separation of components in crude K. parviflora rhizome extracts into fractions was performed by column chromatography followed by chemical characterisations of the adaptogenic-active extract and fraction using nuclear magnetic resonance spectroscopy (NMR) and gas chromatography–mass spectrometry (GC–MS).
2. Materials and methods 2.1. Plant materials Rhizomes of K. parviflora Wall ex Baker Phurua-10 (Roomkraw) were collected in Phurua, Loei Province, in northeastern Thailand in June 2006. Voucher herbarium specimens, QBG No. 27634, of the plant were identified and deposited at the Queen Sirikit Botanic Garden, Mae Rim, Chiang Mai, Thailand.
2.2. Extraction Rhizomes of K. parviflora were shade dried and agitated using a blender until becoming powder. The sample was separated into two sets. For set 1, 100 g of sample were macerated with 250 ml of hexane for 3 days. The solution was subsequently filtered and the residue was re-extracted with hexane (2 250 ml). Then, the residue was extracted again in the same way as above with chloroform and methanol, respectively. All filtrates of the hexane, chloroform, and methanol fractions were collected and evaporated to obtain hexane crude extract (KPHE), chloroform crude extract (KPCE), and methanol crude extract (KPME) with yields of 6.40%, 2.94%, and 4.33% w/w, respectively. For set 2, the sample was extracted following the method of set 1 except that methanol was replaced by ethanol. All filtrates of the hexane, chloroform, and ethanol fractions were collected and evaporated to obtain hexane crude extract (KPHE), chloroform crude extract (KPCE), and ethanol crude extract (KPEE) with yields of 6.40%, 2.90%, and 5.30% w/w, respectively. All crude extracts were individually lyophilised to give a brown–yellow semi-solid material. All extracts obtained from both sets were dissolved in 10% Tween for further adaptogenic analysis.
2.3. Laboratory animals Adult male Swiss albino mice (weighing 25–30 g) obtained from the Animal Unit, Mahidol University, Salaya, Nakornpathom, Thailand, were used in this study. The animals were kept and maintained in environmentally controlled rooms (22 ± 3 °C and 12 h photo-period) and were maintained on a standard rodent pellet diet (S.W.T. Co., Ltd., Sumutprakran, Thailand) and water. Ethical guidelines for the handling of laboratory animals were followed. 2.4. Adaptogenic test 2.4.1. Adaptogenic activity of KPHE, KPCE, KPME and KPEE The ability of adaptogens has often been assessed using a swimming test for mice (Panossian, Wikman, & Wagner, 1999; Perfumi & Mattioli, 2007; Wagner, Norr, & Winterhoff, 1994). For set 1, mice were divided into five groups, with 10 mice per group. The mice in group 1, the control group, were treated with 10% Tween while mice in groups 2, 3, 4, and 5 were treated with KPHE, KPCE, KPME, and KPEE dissolved in 10% Tween, respectively. Each extract of the same oral 500 mg/kg dose and 10% Tween were given once daily at an interval of 24 h for 25 days. After treatment, all mice were forced to swim individually for 6 min in a beaker 10 cm in diameter and 14 cm tall that contained water at room temperature. The duration of immobility was preliminarily measured over 10 min at day 25. For set 2, mice were divided into five groups of 10 mice per group. The mice in group 1, the control group, and group 2, the active control group, were treated orally with 10% Tween and crude powder of ginseng root, Sigma Cat. No. G7253. Mice in groups 3, 4, and 5 were treated with KPHE, KPCE, and KPEE dissolved in 10% Tween, respectively. Each extract of the same oral doses of 500 mg/kg, ginseng root extract of 100 mg/kg, and 10% Tween were given once daily at an interval of 24 h for 35 days. After treatment, all mice were forced to swim individually for 6 min in a beaker 10 cm in diameter and 14 cm tall that contained water at room temperature. The duration of immobility was measured over 10 min at days 0, 5, 10, 15, 20, 25, 30, and 35. Mean and SEM calculated from results obtained by sets 1 and 2 are shown in Tables 1 and 2, respectively. The data were analysed using one-way analysis of variance (ANOVA) and the Duncan multiple range test. P < 0.05 was considered to be statistically significant. 2.4.2. Adaptogenic activity of fractions obtained from hexane extracts The mice were divided into four groups of 10 mice per group. The mice in group 1, which was the control group, were treated with 10% Tween while mice in groups 2, 3, and 4 were treated with KD 2HH1, KD 2HH2, and KD 2HD dissolved in 10% Tween, respectively. Each extract of the same oral doses of 100 mg/kg and 10% Tween were given once daily at an interval of 24 h for 30 days. After treatment, all mice were forced to swim individually for 6 min in a beaker 10 cm in diameter and 14 cm tall that contained
Table 1 Adaptogenic activity of K. parviflora hexane, chloroform, methanol, and ethanol extracts (KPHE, KPCE, KPME, and KPEE) at concentration of 500 mg/kg in the period of 25 days on mice by forced swimming test. Treatment (500 mg/kg)
Immobility time (s)
Control KPHE KPCE KPME KPEE
83 ± 11 43 ± 8* 60 ± 7 77 ± 9 80 ± 6
Data shown as mean ± SEM, n = 8–10. Differ significantly (P < 0.05).
*
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Table 2 Adaptogenic activity of K. parviflora hexane, chloroform, and ethanol extracts (KPHE, KPCE, and KPEE) at concentration of 500 mg/kg and ginseng root crude powder (GP) at concentration of 100 mg/kg on mice by forced swimming test at the day of 0, 5, 10, 15, 20, 25, 30, and 35. Treatment (500 mg/kg)
Control KPHE KPCE KPEE GP
Immobility time (s) at day 0
5
10
15
20
25
30
35
74.20 ± 5.55 68.64 ± 6.11 67.40 ± 6.37 71.49 ± 4.24 70.07 ± 4.64
90.02 ± 5.27 79.17 ± 5.37 86.07 ± 6.87 93.27 ± 6.43 88.41 ± 5.64
105.77 ± 4.58 102.14 ± 4.56 107.12 ± 4.60 106.52 ± 5.27 99.93 ± 4.98
124.91 ± 4.40 118.02 ± 4.78 122.40 ± 4.12 125.05 ± 4.65 112.74 ± 5.22
133.19 ± 8.95 89.76 ± 8.49* 136.74 ± 7.63 127.96 ± 8.37 88.78 ± 7.82*
146.61 ± 7.06 74.01 ± 7.61* 137.06 ± 6.78 135.55 ± 4.95 86.08 ± 8.37
143.75 ± 8.04 57.05 ± 7.56* 147.05 ± 3.68 130.66 ± 8.68 121.43 ± 6.81
137.31 ± 7.99 73.76 ± 7.47* 146.19 ± 9.79 164.85 ± 5.47 129.01 ± 8.83
Data shown as mean ± SEM; n = 8–10. Differ significantly (P < 0.05); KPHE, KPCE, KPEE, and GP=K. parviflora hexane, chloroform, ethanol extracts and ginseng root crude powder.
*
Table 3 Adaptogenic activity of the three fractions separated from K. parviflora hexane extracts (KPHE) at concentration of 100 mg/kg on mice by forced swimming test at day of 0, 5, 10, 15, 20, 25 and 30. Treatment (100 mg/kg)
Control KD 2HH1 KD 2HH2 KD 2HD
Immobility time (s) at day 5
10
15
20
25
30
110.64 ± 9.83 116.37 ± 12.40 123.31 ± 10.58 145.30 ± 9.99
119.88 ± 13.38 111.26 ± 15.05 119.50 ± 12.99 146.44 ± 11.97
151.51 ± 12.37 135.28 ± 14.04 135.38 ± 12.03 166.23 ± 14.03
158.95 ± 12.49 141.88 ± 13.24 129.28 ± 11.48 160.39 ± 4.60
155.46 ± 15.79 158.06 ± 8.17 122.13 ± 11.01* 157.92 ± 6.75
166.38 ± 14.95 150.75 ± 10.28 126.38 ± 12.94* 167.41 ± 12.99
Data shown as mean ± SEM; n = 8–10. Differ significantly (P < 0.05); KD 2HH1, fat fraction; KD 2HH2. terpenoids-rich fraction; and KD 2HD, flavonids-rich fraction.
*
water at room temperature. The duration of immobility was measured over 10 min at days 0, 5, 10, 15, 20, 25, and 30. Mean and SEM were calculated as shown in Table 3. The data were analysed using one-way analysis of variance (ANOVA) and the Duncan multiple range test. P < 0.05 was considered to be statistically significant.
source and quadrupole temperatures were set at 230 and 150 °C, respectively. Identification of individual components in the extract was done by comparing their mass spectra with the reference mass spectra in the Wiley Registry™ 8th Ed. and NIST 2008 databases. This was supported by the data of linear temperature programme retention indices (LRI) calculated from retention times of the added C8 to C22 n-paraffin hydrocarbon mixtures.
2.5. Column chromatography (CC) Twenty-eight grammes of the hexane extract of K. parviflora rhizomes were subjected to CC over silica gel. The crude extract was first suspended in hexane, and the mixture was filtered to give a hexane-soluble part and a residue (22.5 g). The residue contained, as indicated by 1H NMR, mainly flavonoids and fatty acids, and a preliminary activity screening revealed that this residue did not possess adaptogenic activity. The hexane-soluble part was separated by silica gel CC, eluted with a gradient of dichloromethaneethyl acetate (80% dichloromethane-ethyl acetate to 50% dichloromethane-ethyl acetate), acetone, and methanol to provide three fractions containing 3.54 g of fraction KD 2HH1, 1.02 g of fraction KD 2HH2, and a trace amount of fraction KD 2HD. 2.6. Chemical characterisation of adaptogenic-active compounds of KD 2HH2 fraction 2.6.1. Gas chromatography–mass spectrometry (GC–MS) The adaptogenic active fraction KD 2HH2 was analysed for its chemical constituents using a Hewlett Packard model HP6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with an HP-5MS (5% phenyl-polymethylsiloxane) capillary column (30 m 0.25 mm i.d., film thickness 0.25 lm; Agilent Technologies, USA) interfaced to an HP model 5973 mass-selective detector. The oven temperature was initially held at 120 °C and then increased by 2 °C/min to 250 °C. The injector and detector temperatures were 250 and 280 °C, respectively. Purified helium was used as the carrier gas at a flow rate of 1 ml/min. EI mass spectra were collected at 70 eV ionisation voltages over the range of m/ z 29–300. The electron multiplier voltage was 1150 V. The ion
2.6.2. Nuclear magnetic resonance (NMR) 1 H NMR spectra were recorded on a Bruker AM 400 (400 MHz for 1H) (Bruker, Germany), and samples were dissolved in CDCl3 with Si(CH3)4 as an internal standard. 3. Results and discussion 3.1. Effect of extracts and fractions on swim endurance Adaptogenic activities of all crude extracts obtained by extraction in set 1 on the 25-day forced swimming test of mice are shown in Table 1. In this table the immobility times of the groups treated with KPCE, KPME and KPEE are not significant compared to the control group while mice treated with KPHE show significantly shorter immobilization times than those treated with the other extracts. The duration of immobility times at days 0, 5, 10, 15, 20, 25, 30, and 35 of mice treated with crude extracts obtained by extraction in set 2 are shown in Table 2. In this table the immobility times of the groups treated with KPCE and KPEE are not significantly shorter than those of the control group. However, the table shows a significantly shorter immobilization times in mice treated with KPHE than those of the control group at days 20, 25, 30, and 35. According to this significantly shorter immobilization time of KPHE, this extract was subjected to CC for further investigation of its chemical components. Three fractions were isolated consisting of KD 2HH1, KD 2HH2, and KD 2HD. All fractions were tested for their adaptogenic activities. The adaptogenic effects of these fractions at a concentration of 100 mg/kg on mice by the forced swimming test at days 0, 5, 10, 15, 20, 25, and 30 are shown in Table 3. The table shows no significant difference in body weight be-
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tween mice in the control group and the other groups but it does show significant differences in the immobility times between the control group and treatment groups. The immobilization times decreased considerably in the groups treated with KD 2HH2 at days 25 and 30, with the minimal time of 122.13 ± 11.01 s and 126.38 ± 12.94 s compared to the control group, which showed a minimal time of 155.46 ± 15.79 s and 166.38 ± 14.95 s, respectively, for the same days. On the other hand, the immobility times of the groups treated with KD 2HD and KD 2HH1 were not significantly shorter than those of the control group. Dead mice were operated on after the experiment was concluded. Operation results showed there were no significant differences in the average weight of hearts, livers, and adrenal glands of the group treated with KD 2HH2 compared to weight of the same glands of the control group. In this study, the kidney weight in the group treated with KD 2HH1 was significantly greater compared to that of the groups treated with KD 2HD, KD 2HH2, and the control group. As can be seen, KD 2HH2 can be attributed to less exhaustion of the swimming mice, with no effect on heart, liver, kidney, and adrenal gland weight. Different extracts of a dose of 100 mg/kg were given orally once daily every 24 h for 30 days. The results are shown in Table 4. Shorter immobilization times occurred on mice treated with KD 2HH2 and crude powder of ginseng root compared to those of the control group at day 25. This indicated that the adaptogenic activity of KD 2HH2 was comparable to that of the ginseng root and was higher than that of the control group. Moreover, there was no significant difference in body weight between the mice of the control group and the mice treated with both KD 2HH2 and crude ginseng root powder. It was found that both extracts decreased the immobilization time of mice at days 15, 20, and significantly at days 25 and 30 compared to those of the control group. Moreover, no significant differences of body, heart, liver, kidney, and adrenal gland weights were found for mice treated with KD 2HH2 and crude ginseng root powder. Thus, the KD 2HH2 fraction was considered to be the potent and non-toxic adaptodenic-active fraction that showed similar results as those obtained from the same oral doses of crude ginseng root powder on mice for 30 days. The many studies on chemical characterisation and pharmacological investigations for organ specific treatment have found that plants are still an ideal choice as adaptogens. Such plant adaptogens are Panax spp. (Lewis, Zenger, & Lynch, 1983), Caesalpinia bonduc (Kannur et al., 2006), Hippophae rhamnoides (Seggu et al., 2006), Evolvulus alsinoides (Siripurapu et al., 2005), Trichopus zeylanicus (Singh et al., 2005), Bacopa monniera (Rai et al., 2003), and Withania somnifera (Bhattacharya & Muruganandam, 2003). Most adaptogenic-active components appear to induce a state of nonspecific increase of resistance of the organism to diverse aversive assaults that threaten internal homoeostases and which improve physical endurance for doing work even in adverse circumstances and in difficult environmental conditions. These components act as more preventive than curative when body resistance has been decreased by such things as chronic illness, long-term stress and old age (Bhattacharya, Bhattacharya, Bhattacharya, & Chakraborthy,
2000). The results of this study indicate that the KD 2HH2 fraction obtained from the hexane extract of K. parviflora has an adaptogenic property similar to that of crude ginseng root powder. The ginseng root has been recognised as an adaptogenic plant since ancient times. The results of this study indicate that the KD 2HH2 fraction from the hexane extract of K. parviflora rhizomes has a protective action against exhaustion on swimming mice and can possibly be used as an alternative adaptogenic medicinal herb.
3.2. Identification of adaptogenic-active components in HD 2HH2 fraction Three fractions of KPHE; KD 2HH1, KD 2HD, and KD 2HH2, were obtained after separation by CC. The 1H NMR spectra showed that fractions KD 2HH1 and KD 2HD were rich in fat and flavonoids, respectively, while fraction KD 2HH2, which was responsible for the observed adaptogenic activity, contained terpene constituents. The chemical compositions of the KD 2HH2 fraction obtained from K. parviflora rhizomes were characterised by GC–MS and NMR techniques. Components of the hexane extract and the KD 2HH2 fraction identified by GC–MS are shown in Table 5. All identified components were in groups of monoterpenes and sesquiterpenes, as well as their derivatives. Thirty-two volatile constituents were identified in the hexane extract of K. parviflora rhizomes, these representing 94.95% of volatile components in the extract. The dominant components of the hexane extract were germacrene D (23.97%), borneol (10.24%), b-pinene (8.60%), camphene (7.62%), a-copaene (7.23%), and linalool (6.40%). Table 5 also shows some minor constituents, including a-pinene, E-caryophyllene, b-elemene, b-selinene, and d-amorphene. Fourteen constituents representing 94.04% of the KD 2HH2 fraction were identified. The principal components of these were germacrene D (21.27%), belemene (19.61%), a-copaene (13.91%), E-caryophyllene (10.97%), Z-muurola-4(14),5-diene (5.97%), and a-humulene (4.56%). The KD 2HH2 fraction also had some minor constituents, these being d-amorphene, germacrene A, b-selinene, c-cadinene, and b-bourbonene. 1H NMR spectra have proven useful in identification of herbal crude extracts (Bilia, Bergonzi, Lazari, & Vincieri, 2002; Pauli, Jaki, & Lankin, 2005; Rivero-Cruz, Rivero-Cruz, Rodríguez, CerdaGarcía-Rojas, & Mata, 2006). The 1H NMR spectrum (CDCl3) of the crude hexane extract of K. parviflora rhizomes showed that fat, or triglycerides, flavonoids, and germacrene D were the major constituents. Previous chemical investigation of K. parviflora rhizomes revealed that flavonoids were major secondary metabolites in the rhizomes (Yenjai et al., 2004). However, Rivero-Cruz and coworkers (2006) reported characteristic chemical shifts (dH) in the region of 7.10–4.20 ppm of particular terpenoids, including chamazulene (dH 6.98 ppm), b-caryophyllene (dH 4.95 ppm), germacrene D (dH 5.78 ppm), bicyclogermacrene (dH 4.33 ppm), and b-eudesmol (dH 4.48 ppm). In this study, the 1H NMR spectrum of a crude hexane extract of K. parviflora rhizomes clearly showed the signal at dH 5.78 ppm of germacrene D, and this terpene was revealed 23.97%
Table 4 Adaptogenic activity of K. parviflora active fraction (KD 2HH2) and ginseng root crude powder (GP) at concentration of 100 mg/kg on mice by forced swimming test at day 0, 5, 10, 15, 20, 25, and 30. Treatment (100 mg/kg)
Control KD 2HH2 GP
Immobility time (s) at day 0
5
10
15
20
25
30
90.72 ± 3.68 92.35 ± 3.96 89.47 ± 4.12
115.20 ± 5.15 107.29 ± 4.01 117.32 ± 5.58
132.57 ± 4.46 132.70 ± 3.58 136.84 ± 3.12
154.00 ± 5.48 141.48 ± 4.07 142.74 ± 3.34
165.38 ± 6.39 150.20 ± 4.16 158.78 ± 4.42
175.91 ± 4.90 156.52 ± 4.84* 145.82 ± 3.68*
191.75 ± 4.61 165.36 ± 4.59* 145.62 ± 4.02*
Data shown as mean ± SEM; n = 8–10. * Differ significantly (P < 0.05); KD 2HH2 and GP = K. parviflora active fraction and ginseng root crude powder.
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Table 5 GC–MS structural assignment and relative peak area percent of the identified components of hexane extract and KD 2HH2 fraction obtained from K. parviflora rhizomes. No.
Compound
LRIa
LRIb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Tricyclene a-Pinene Camphene Sabinene b-Pinene Myrcene a-Phellandrene p-Cymene Limonene 1,8-Cineole b-E-Ocimene Terpinolene Linalool Borneol Terpinen-4-ol a-Terpineol Isobornyl acetate Methyl-anthranilate a-Copaene b-Bourbonene b-Cubebene b-Elemene E-Caryophyllene b-Cedrene a-Humulene Z-Muurola-4(14),5-diene E-Cadina-1(6),4-diene Germacrene D b-Selinene Germacrene A c-Cadinene d-Amorphene
928 941 962 991 999 1006 1020 1035 1038 1041 1063 1086 1101 1174 1182 1198 1282 1344 1373 1381 1384 1388 1416 1427 1453 1469 1473 1479 1492 1504 1511 1516
924 925 940 972 968 982 999 1024 1027 1030 1050 1079 1098 1164 1171 1188 1270 1333 1366 1372 1378 1382 1416 1420 1443 1458 1464 1472 1477 1491 1504 1511
% RA HEX
KD 2HH2
0.12 5.65 7.62 0.10 8.60 0.58 0.11 0.07 1.36 1.26 0.08 0.21 6.40 10.24 0.22 0.26 0.31 1.88 7.23 0.50 0.59 3.58 4.94 0.17 0.75 1.24 0.56 23.97 2.42 1.44 0.16 2.33
– – – – – – – – – – – – – – – – – – 13.91 1.36 0.54 19.61 10.97 0.54 4.56 5.97 0.54 21.27 3.92 3.93 2.96 3.96
%RA, Percentage of relative peak area; HEX, Hexane extract of K. parviflora rhizomes; KD 2HH2, KD 2HH2 fraction. a Linear temperature programme retention index. b Linear temperature programme retention index from Literatures (Adams, 1998).
of the total amount of volatile constituents in the crude extract detected by GC–MS technique (Table 5). The 1H NMR spectrum of the active fraction KD 2HH2 showed that germacrene D was major, together with some unidentified terpenes resonated at dH 0.7– 2.1 ppm. These terpenes were subsequently separated and identified by the GC–MS technique (Table 5) with germacrene D showing the highest amount of 21.27%.
4. Conclusions This study has shown that terpenoids might increase the adaptogenic activity in mice and that the terpene fraction has comparable adaptogenic activity as that of crude ginseng root powder. This research is supported by the study of Willard (1990), who reported that terpenes give Reishi mushroom an adaptogenic quality, providing protection from a wide range of biological, environmental, and social stresses. Reishi mushrooms have been used in Western medicine to increase resistance by stimulating the immune system and also by normalising, or modulating, immune response. Eugenol and caryophyllene were considered as the most imperative adaptogenic agents present in Ocimum sanctum essential oil reported by Prakash and Gupta (2005). These terpenes were very effective in lowering corticosterone levels that are the main cause of stress. In addition, germacrence D and caryophyllene were found to provide adaptogenic properties in Thymus serpyllum and Humulus lupulu essential oils, respectively, as reported by Pozharitskaya et al.
(2008). Rhodiola rosea essential oil had adaptogenic properties with monoterpene alcohols as the major constituents, as reported by Morgan and Bone (2005). In this study, adaptogenic activities could be related to the presence of various types of major terpenoids in the KD 2HH2 fraction, such as germacrene D, b-elemene, a-copaene, and E-caryophyllene.
Acknowledgments Financial support from the Agricultural Research Development Agency is gratefully acknowledged. We thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education for its support of the GC instrument and Sanya Sureram for technical assistance.
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