Chemical Constituents from the Fruits of Amomum kravanh and Their Role in Activating Alcohol Dehydrogenase

Alcoholism is a worldwide health problem, and diseases caused by alcoholism are killing people every year. Amomum kravanh is a traditional Chinese medicine used to relieve hangovers. However, whether its bioactive components improve alcohol metabolism is not clear. In this study, ten new (amomumols A-J, 1–10) and thirty-five known (11–45) compounds were isolated from the fruits of Amomum kravanh by an activity-guided separation. Ten novel compounds were identified as four sesquiterpenoids (1–4), three monoterpene derivatives (5–7), two neolignans (8, 9), and a novel norsesquiterpenoid (10) with a new C14 nor-bisabolane skeleton. Their structures were determined by the comprehensive analysis of high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) calculation. The effects of all isolated compounds on the activity of alcohol dehydrogenase were evaluated in vitro, and it was found that eight compounds (11, 12, 15, 18, 26, and 36–38) exhibited significant activation effects on the alcohol dehydrogenase at 50 μM.


Introduction
Drinking culture is shared by all nationalities in the world. However, regarding public health, heavy drinking has become a thorny global healthcare problem [1]. As the leading metabolic site of alcohol, the liver is responsible for most of the damage caused by alcohol metabolism. Heavy drinking results in a high incidence of alcoholic liver diseases (ALD), including fatty lesions, liver fibrosis, cirrhosis, and even acute and chronic hepatitis and liver cancer [2,3]. In addition to the liver, the brain, heart, and gastrointestinal tract will be partially damaged by excessive alcohol consumption [4][5][6]. To date, there is no specific drug to help people eliminate alcoholism, and few drugs, such as metadoxine, interleukin-22 analogues, and interleukin-1β antagonists, are available for ALD [7]. In Asia, including China, Japan, and Korea, botanical medicines are traditionally used to prevent and treat alcohol-related diseases [8,9].
White cardamom, the fruits of Amomum kravanh Pierre ex Gagnep and Amomum compactum Soland ex Maton (Zingiberaceae), is used worldwide as a spice in cooking to change the taste of food. In China, cardamom is used as a spice as well as herbal medicine to treat stomach and digestive diseases. Chinese people also use cardamom to promote alcohol metabolism, which was recorded in many ancient traditional medicine books [10]. However, the ingredients and mechanism of cardamom to promote alcohol metabolism have not been studied deeply. A. kravanh is a tropical plant native to Cambodia and Thailand. Previous phytochemical investigations showed that the main components of A. kravanh are volatile oil, diterpenoids, flavonoids, steroids, diarylheptane, and lignans [10][11][12].
In this study, we tried to find bioactive compounds from the fruit of A. kravanh that have the potential to help alcohol metabolism. An in vitro alcohol dehydrogenase activity (ADH) assay was used for the activity-guided isolation. It led to the isolation of 10 new (1-10) and 35 known (11-45) compounds ( Figure 1) from the fruits of A. kravanh and the identification of 8 bioactive compounds (11,12,15,18,26, and 36-38) with alcohol dehydrogenase activation. Herein, the isolation, structural elucidation, and bioassay of 10 new (1-10) and 35 known  compounds from the fruits of A. kravanh are reported.
In this study, we tried to find bioactive compounds from the fruit of A. kravanh tha have the potential to help alcohol metabolism. An in vitro alcohol dehydrogenase activity (ADH) assay was used for the activity-guided isolation. It led to the isolation of 10 new (1-10) and 35 known (11-45) compounds ( Figure 1) from the fruits of A. kravanh and the identification of 8 bioactive compounds (11,12,15,18,26, and 36-38) with alcohol dehy drogenase activation. Herein, the isolation, structural elucidation, and bioassay of 10 new (1-10) and 35 known  compounds from the fruits of A. kravanh are reported.

Results and Discussion
The fruits of A. kravanh (10 kg) were extracted with 80% EtOH under reflux and then partitioned successively with petroleum ether (PE), EtOAc, and n-BuOH. The effects of these extracts on alcohol dehydrogenase activation were screened. Results showed that PE and EtOAc fractions could significantly activate alcohol dehydrogenase ( Figure S103). Therefore, PE and EtOAc fractions were subjected to silica gel column chromatography and further purified by repeated MPLC and HPLC to obtain four new sesquiterpenoids (1)(2)(3)(4), three new monoterpene derivatives (5-7), two new neolignans (8,9), a novel norsesquiterpenoid (10), and thirty-five known compounds   (Figure 1).
Compound 4 was obtained as yellowish oil with a molecular formula C 15 (Table 1) were closely comparable to those of (2R,6S)-2,6-dihydroxylhumla-9E,3(12),7(13),9-triene [20], except that a hydroxy group was replaced by a ketone in 4. The planar structure of 4 was then fully elucidated by 2D NMR data ( Figure 2). It was found that the positive cotton effect at 230 nm in the ECD of 4 matched well with those of the calculated ECD of 6S-4. Therefore, the absolute configuration of 4 was assigned to be 6S and named amomumol D.

of H-3/H-4/H-5/H-6, H-5/H-7/H-8(H-9). 1 H and 13 C NMR data (
Amomumol H (8) was obtained as an amorphous powder, and its molecular formula was established to be C 22 (Table 3) were closely similar to alismaines A and B, two diphenylpropanoid ethers from Alismatis Rhizoma [23]. Two hydroxymethyl groups at C-9 and C-9 and a methoxy group C-7 in alismaines A and B were replaced by two methyl groups and a hydroxy group in 8.  [23,24]. The opposite optical rotation and ECD curve of 8 compared with those of alismaine A (7S,8R) suggested that the absolute configuration of 8 is (7R,8S).  The molecular formula C22H28O6 of 9 has one less oxygen atom than that of 8. The 1 H and 13 C NMR data (Table 3) of 9 were very similar to those of 8. The difference between the two compounds is that 9 possesses methylene at C-7 instead of an oxygenated methine of 8. The structure of 9 was then fully elucidated by 2D NMR data analysis ( Figure 6). Compound 9 has only one chiral carbon at C-8, but no cotton effect in the ECD spectrum of 9 was observed. Therefore, 9 was considered to be a racemate and named amomumol I.  The molecular formula C22H28O6 of 9 has one less oxygen atom than that of 8. The 1 H and 13 C NMR data (Table 3) of 9 were very similar to those of 8. The difference between the two compounds is that 9 possesses methylene at C-7 instead of an oxygenated methine of 8. The structure of 9 was then fully elucidated by 2D NMR data analysis ( Figure 6). Compound 9 has only one chiral carbon at C-8, but no cotton effect in the ECD spectrum of 9 was observed. Therefore, 9 was considered to be a racemate and named amomumol I.  The molecular formula C22H28O6 of 9 has one less oxygen atom than that of 8. The 1 H and 13 C NMR data (Table 3) of 9 were very similar to those of 8. The difference between the two compounds is that 9 possesses methylene at C-7 instead of an oxygenated methine of 8. The structure of 9 was then fully elucidated by 2D NMR data analysis ( Figure 6). Compound 9 has only one chiral carbon at C-8, but no cotton effect in the ECD spectrum of 9 was observed. Therefore, 9 was considered to be a racemate and named amomumol I. The molecular formula C 22 H 28 O 6 of 9 has one less oxygen atom than that of 8. The 1 H and 13 C NMR data (Table 3) of 9 were very similar to those of 8. The difference between the two compounds is that 9 possesses methylene at C-7 instead of an oxygenated methine of 8. The structure of 9 was then fully elucidated by 2D NMR data analysis ( Figure 6). Compound 9 has only one chiral carbon at C-8, but no cotton effect in the ECD spectrum of 9 was observed. Therefore, 9 was considered to be a racemate and named amomumol I.
Compound 10 (amomumol J) has a molecular formula C 14 H 22 O 2 calculated by its HRESIMS ion at m/z 223.1697 [M + H] + . The 1 H and 13 C NMR data (Table 1) (Figure 6). As with other bisabolene-type sesquiterpenoids, the ring and side train moieties were connected by C4-C7 bond. In the 13 C NMR spectrum of 10, the carbon resonances corresponding to the 2-methyl-3-hydroxyhept-1-ene side train showed a pair of resonances for each carbon, which may be caused by the different configurations of C-10. To separate the possible isomer of 10, we tried different HPLC conditions, including the chiral column. However, compound 10 always showed a peak in different HPLC chromatographies. Next, we modified the hydroxyl group at C-10 of 10 to obtain the acetate of 10 (10a). In the 13 C NMR spectrum of 10a, the split carbons were still observed. Unfortunately, the separation of 10a was not successive. Although the configuration of 10 has not been determined, compound 10 is the first example of norsesquiterpenoid with a new C14 nor-bisabolane skeleton.

In Vitro Alcohol Dehydrogenase (ADH) Promoting Activity of Isolated Compounds
Inspired by the results of the activity screening of the extracts, we evaluated the effects of 45 purified compounds on ADH enzyme activity by in vitro assays. All compounds were first screened using 50 µM of test compounds, and then concentration-dependent experiments (25,50, and 100 µM) were performed on compounds with enhancing ADH enzyme activity. It was found that 11, 12, 15, 18, 26, and 36-38 showed a concentrationdependent increase in ADH enzyme activity (Figure 7). These eight compounds belong to three steroids (12,15, and 18), one flavonoid (27), one sesquiterpenoid (11), and three diterpenoids (36)(37)(38). Kravanhin B (36), a hemanthane-type diterpenoid, exhibited the most enhancing ADH enzyme activity compared to other compounds. Steroids with the peroxo bridge group showed the potential to activate ADH more than other steroids. This is the time the effects of these known compounds on ADH activation have been reported. These results suggested that the effect of A. kravanh on improving alcohol metabolism may come from the impact of the combination of multiple components. ene side train showed a pair of resonances for each carbon, which may be caused by the different configurations of C-10. To separate the possible isomer of 10, we tried different HPLC conditions, including the chiral column. However, compound 10 always showed a peak in different HPLC chromatographies. Next, we modified the hydroxyl group at C-10 of 10 to obtain the acetate of 10 (10a). In the 13 C NMR spectrum of 10a, the split carbons were still observed. Unfortunately, the separation of 10a was not successive. Although the configuration of 10 has not been determined, compound 10 is the first example of norsesquiterpenoid with a new C14 nor-bisabolane skeleton.

In Vitro Alcohol Dehydrogenase (ADH) Promoting Activity of Isolated Compounds
Inspired by the results of the activity screening of the extracts, we evaluated the effects of 45 purified compounds on ADH enzyme activity by in vitro assays. All compounds were first screened using 50 µM of test compounds, and then concentration-dependent experiments (25,50, and 100 µM) were performed on compounds with enhancing ADH enzyme activity. It was found that 11, 12, 15, 18, 26, and 36-38 showed a concentration-dependent increase in ADH enzyme activity (Figure 7). These eight compounds belong to three steroids (12, 15, and 18), one flavonoid (27), one sesquiterpenoid (11), and three diterpenoids (36)(37)(38). Kravanhin B (36), a hemanthane-type diterpenoid, exhibited the most enhancing ADH enzyme activity compared to other compounds. Steroids with the peroxo bridge group showed the potential to activate ADH more than other steroids. This is the time the effects of these known compounds on ADH activation have been reported. These results suggested that the effect of A. kravanh on improving alcohol metabolism may come from the impact of the combination of multiple components.

Plant Material
The dried fruit of A. kravanh was purchased from Guangdong Kangmei Pharmaceutical Co., Ltd., Puning, China, in September 2021. The samples were kept in the State Key Laboratory of Quality Research in Chinese Medicines (Macau University of Science and Technology) after being identified by Prof. G.-Y. Zhu.

Extraction and Isolation
The dried fruits power of A. kavanh (10 kg) were extracted by 80% EtOH under reflux for 1 h each time (4 × 20 L). The solvent was removed under reduced pressure to obtain the crude extract (300 g), which was suspended in water and then partitioned successively with PE, EtOAc, and n-BuOH to obtain PE extract (80 g), EtOAc extract (57 g), and n-BuOH (17 g).

Acetylation Reaction of Compound 10
This acetylation reaction used the traditional pyridine-acetic anhydride method to acylate the acetyl group to the OH-10 of 10. Compound 10 (4 mg) was dissolved in 150 µL of pyridine, and then 50 µL of acetic anhydride was added to react at room temperature for 3 h. The solvent was recovered to obtain compound 10a (4.5 mg).

Assay for ADH-Promoting Activity In Vitro
A modified traditional Valle-Hoch method was used to measure ADH activity [43]. Briefly, 10 µL of 27 mM NAD + solution, 10 µL of ADH (0.1 mg/mL) solution, and 10 µL of different concentrations of test compounds (metadoxine as a positive control) were added into 60 µL of 32 mM sodium pyrophosphate buffer (pH 8.8). The negative control group used 10 µL buffer instead of the sample solution. After incubating at room temperature for 30 min, 10 µL of 5% alcohol was added to the mixture for 5 min. The absorbances at 340 nm wavelength were then taken using a microplate reader.

Conclusions
To identify the bioactive compounds from A. kravanh, we used in vitro assay to evaluate the ADH-promoting activity of the PE, EtOAc, and n-BuOH fractions of the extract of the fruits of A. kravanh and found that the PE and EtOAc fractions showed ADH enhancing activity. The following phytochemical investigation resulted in the isolation of four new sesquiterpenoids (1-4), three new monoterpene derivatives (5-7), two new neolignans (8 and 9), a novel norsesquiterpenoid (10), and thirty-five known compounds. The bioassay results showed that compounds 11, 12, 15, 18, 26, and 36-38 significantly enhanced alcohol dehydrogenase activity in a dose-dependent manner. These results give a new insight into the chemical diversity and the potential usage in the hangover cure of A. kravanh.