PHENOLIC COMPOUNDS FROM INDONESIAN WHITE TURMERIC ( CURCUMA ZEDOARIA ) RHIZOMES

Objective: The aim of the present study is to isolate phenolic compounds from Curcuma zedoaria rhizomes grown in Bogor, West Java, Indonesia, which will enrich phytochemical information from this plant. Methods: C. zedoaria rhizomes were macerated in methanol then followed by increasing polarity partitions with n -hexane, ethyl acetate (EtOAc), and methanol, respectively. EtOAc fraction was further fractionated using various chromatography techniques to yield two isolated fractions, Z1 and Z2. These two isolated fractions were then characterized to determine their compound structures. Results: Fourier Transform-InfraRed (FTIR), Ultraviolet-Visible (UV-Vis), and Liquid Chromatography Mass Spectrometry tandem Mass Spectrometry LC-MS/MS spectral data, Z1 fraction was elucidated as curcuminoid derivative, that is, dimethoxycurcumin (DiMC, 1 ), while Z2 fraction was yielded as a mixture consisted of flavonoid and coumarin derivatives, 3,5,7-trihydroxy-4’-methoxyflavone (kaempferide, 2 ) and 7-methoxy coumarin (herniarin, 3 ). Conclusion: This study reveals useful information regarding phenolic constituents of Indonesian C. zedoaria rhizomes. Further research needs to be carried out to purify other compounds contained and to conduct bioactivity assays.


INTRODUCTION
In recent years, natural products derived from terrestrial plants, animals, microorganisms, and marine organisms play an important role in traditional medicines [1]. Curcuma zedoaria, also known as temu putih, white turmeric, zedoaria, or gajutsu, is belonging to Zingiberaceae family and close relative to Curcuma longa. This plant has dark orange-fleshed tubers similar to C. longa. This plant is indigenous to Bangladesh, Sri Lanka, India and is also widely cultivated in China, Japan, Brazil, Nepal, and Thailand [2]. Its rhizomes are commonly consumed traditionally as medicine in Asia for curing stomach diseases, toothache, blood stagnation, leucoderma, tuberculosis, enlargement of spleen, and for promoting menstruation, while the roots are usually used in the treatment of flatulence, dyspepsia, cold, cough, fever, and infections [3]. Phytochemical investigation of this plant showed that C. zedoaria is a rich source of essential oils, terpenoids, and curcuminoids [4,5]. C. zedoaria was also reported to have wide range of pharmacological activities such as antimicrobial and antifungal, anti-amoebic, larvicidal effect, antinociceptive, analgesic, antiallergic, antiulcer, anti-inflammatory, hemagglutinating, antimutagenic, anticancer, and hepatoprotective [5,6].
Having predominant of terpenoids, C. zedoaria rhizomes capture scholars' attention to investigate novel terpenoids or essential oils from this plant. Two diterpenes, curcuzedoalide and curcuminol D, were obtained from C. zedoaria cultivated in South Korea [7]. Curdione and curcumol belonging to sesquiterpenes had been isolated previously from essential oils of Chinese C. zedoaria rhizomes [8]. From methanol extract of C. zedoaria rhizomes, purchased from Kyoungdong Herbal Market in Seoul, five sesquiterpenes had been reported such as isoprocurcumenol, germacrone, curzerenone, curcumenol, and curcuzedoalide [9]. Moreover, since known as terpenoids-rich plant, this plant, together with other species Curcuma, had been profiled for distinguishing Indian Curcuma species based on its non-polar terpenes contains [10]. Nevertheless, there are still limited studies on phenolic compounds contained from C. zedoaria rhizomes. Align with our interest in a phytochemical investigation of Indonesian Curcuma [11][12][13][14][15][16][17], in the present study, we conduct phenolic isolation and characterization from methanol crude extract of C. zedoaria rhizomes grown in West Java, Indonesia. and de-solvation temperature of 350°C. Chemicals used for isolation were in both technical (CV. Satya Darmawan) and pro analysis (Merck) grades, such as, methanol (MeOH), n-hexane, ethyl acetate (EtOAc), dichloromethane (DCM), chloroform (CHCl 3 ), and acetone.

Collection of plant material
C. zedoaria, Rosc. rhizomes were collected and identified from Biopharmaca Research Centre, Institut Pertanian Bogor, West Java, Indonesia.

Isolation of phenolic compounds from C. zedoaria rhizomes
Fresh-harvested C. zedoaria rhizomes were washed, sliced into small pieces, dried, and ground into a fine powder using a powdering mill. The air-dried powdered rhizomes (2.0 kg) were then extracted 3 times with MeOH at room temperature. MeOH crude extract (65 g) was then partitioned with increasing polarity using n-hexane, EtOAc, and MeOH, respectively, afforded 7.17 g n-hexane, 30.59 g EtOAc, and 4.31 g MeOH extracts.

RESULTS AND DISCUSSION
C. zedoaria, belonging to Zingiberaceae family, was selected for the present study by recent literature showed that this species is usually used as traditional medicine and recognized to be a rich source of terpenoids [18,19]. However, relatively little was explored regarding extraction and separation processes of phenolic compounds from C. zedoaria rhizomes. Phenolics investigation of MeOH crude extract of C. zedoaria rhizomes using successive various chromatography techniques resulted in the isolation and characterization of two fractions, that is, Z1 fraction which was identified as dimethoxycurcumin (DiMC, 1) and Z2 fraction which was obtained in a mixture of 3,5,7-trihydroxy-4'-methoxyflavone (kaempferide, 2) and 7-methoxy coumarin (herniarin, 3). Fig. 1 showed the chemical structures of isolated phenolics from C. zedoaria rhizomes which were elucidated using FTIR, UV-Vis, and LC-MS/MS. FTIR spectra of 1 (Z1 fraction) showed characteristic peaks of phenolics at a wavenumber of 3598-3394, 2955, 1510, and 1267 cm −1 indicated O-H, C-H sp 2 , C=C aromatic, and C-O-C ether, respectively. In addition, this compound also showed peaks for C-H sp 3 , C=O, and C=C alkene at a wavenumber of 2928-2860, 1733, and 1604, respectively. Phenolics have an aromatic ring with at least one hydroxyl group [20]. The presence of hydroxyl group (O-H) in the FTIR spectra of 1 is due to keto-enol tautomerization. Moreover, maximum absorbance in UV-Vis spectra of 1 appeared at the wavelength of 263 and 402 nm. A peak at 263 nm indicated benzoyl chromophore, while a peak in the visible region (402 nm) specified as a curcuminoid chromophore proven by the yellow appearance of 1 [21]. To support FTIR and UV-Vis analysis, LC-MS/MS characterization of 1 was recorded. LC chromatogram resulted from the positive ion method showed one dominant peak at the retention time of 12.77 min (75.72%). MS spectra showed the  (Fig. 2).
To the best of our knowledge, this is the first report on isolating compound 1 from MeOH crude extract of Indonesian C. zedoaria rhizomes. This compound belonging to curcuminoids was obtained previously from Indian turmeric species (C. longa Linn.) [22,23]. This compound is an analog of curcumin which is commonly known as synthetic curcumin derivative displayed a wide range of bioactivities such as antiproliferative, antioxidant, anti-inflammatory, and anticancer [24][25][26][27]. Moreover, curcuminoid derivatives are found to be the major compounds in several turmeric species. Bisdemethoxy curcumin had previously reported from Chinese C. zedoaria rhizomes [28].
A mixture of 2 and 3 (Z2 fraction) exhibited typical absorption peaks of phenolics, that is, O-H, C-H sp 2 , C-O-C ether, and C=C aromatic at the wavenumber of 3314-3176, 2959, 1550, and 1262 cm -1 , respectively. Besides, this mixture also showed peaks at the wavenumber of 2927-2857, 1750, and 1616 cm -1 indicated C-H sp 3 , C=O, and C=C alkene, respectively. Furthermore, UV-Vis spectra of the mixture showed the maximum wavelength of 275 and 323 nm belongs to benzoyl and cinnamoyl chromophores, respectively, indicated the characteristic peaks for flavonoid, especially flavonol [32]. Shift reagents were then used to elucidate the substituent in flavonoids [33] and summarized in Table 1.
According to Table 1, the mixture consisted of predominant flavonol having three free hydroxyl groups at positions of C3, C5, and C7. Hydroxyl group, which usually appears at C4' position, appeared as methoxy (-OCH 3 ) substituent. Furthermore, there were no o-dihydroxyl groups in both ring A and B in flavonol. Since the fraction was still in a mixture proven by the appearance of a λ max of 215 nm, LC-MS/MS was carried out to analyze the minor compound. LC chromatogram of the mixture revealed two peaks at the retention time of 6.77 and 9.35 min with the average area of 24.29% and 68.16%, respectively. A peak at 9.35 min was identified as 2 showing the dominant compound in the mixture, while a peak at 6.77 min belonged to 3 based on MS spectra analysis. Compound 3 also showed the cinnamoyl chromophore analyzed using UV-vis. Therefore, this peak in UV-Vis appeared in a simultaneous way with a similar chromophore of compound 2. Moreover, the coumarin derivative 3 was reported to have a strong absorption peak at 200-250 and 300-350 nm [34,35]. MS spectra of a peak at 9. 35 [36][37][38]. Furthermore, compound 3 had been reported previously from Matricaria chamomilla, Zanthoxylum zanthoxyloides, and Alpinia calcarata (Zingiberaceae) [39][40][41].

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this work. The funders had no role in the design of the study; in the data collection, analyses, or interpretation; in the writing of the manuscript; or in the decision to publish the results.