PHYTOCHEMICAL STUDY OF INDONESIAN CURCUMA ROTUNDA RHIZOME AND ITS ANTIOXIDANT ACTIVITY TOWARDS 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH)

PHYTOCHEMICAL STUDY OF INDONESIAN CURCUMA ROTUNDA RHIZOME AND ITS ANTIOXIDANT ACTIVITY TOWARDS 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH)

D. U. C. Rahayu ¹, D. Nurfadhilah ² and P. Sugita ^{* 2}

Department of Chemistry ¹, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Kampus UI Depok-16424, West Java, Indonesia.

Department of Chemistry ², Faculty of Mathematics and Natural Sciences, Jalan Tanjung, Kampus Institut Pertanian Bogor (IPB), Dramaga Bogor 16680, Bogor, West Java, Indonesia.

ABSTRACT: Curcuma rotunda, locally known as temu kunci, has been reported containing flavonoids in its rhizome that exhibit antioxidant activity. This study aimed to isolate secondary metabolites from the polar fraction of acetone extract from C. rotunda rhizome and to determine their antioxidant activities. The secondary metabolites were isolated using various chromatography methods yielded two fractions. Identification of both isolated fractions using LC-MS showed a dominant peak on the retention time of 13.30 and 11.30 minutes suspected pinostrobin (chalcone) and pinocembrin (flavanone), respectively. Antioxidant activity towards DPPH of crude extract showed moderate activity with IC₅₀ of 125.75 ppm. However, the antioxidant activities from both isolated fractions could not be determined due to less than 50% of its inhibition percentage. It can be suggested that other specific phenolic compounds in C. rotunda corresponding to its radical scavenging activity.

Curcuma rotunda, Pinostrobin, Pinocembrin, Antioxidant, DPPH

INTRODUCTION: Curcuma rotunda (Zingiberaceae) is widely used as a spice in some Asian countries such as Indonesia, Malaysia, and Thailand. Commonly known as temukunci in Indonesia, C. rotunda can be used to cure diseases such as stomach pain and discomfort, viral and bacterial infection, and diuretic agent, traditionally ¹. It commonly used by Indonesian women after child birth under the traditional name ‘jamu.’ The rhizomes of this plant are effective against inflammatory diseases such as wounds, swellings, and tooth and gum diseases ².

Several medicinal properties such as antimicrobial, antibacterial, antioxidant, antiulcer, antimutagenic, and anticancer can be attributed to different compounds isolated from different parts of this plant ³.

Chemical studies of C. rotundarhizomereported several monoterpenoids, i.e. geranial and neral; chalcones, i.e. boesen bergin A, boesen bergin B, cardamonin, pinostrobin, panduratin A, and dihydro methoxy chalcone; and flavanones, i.e. pinocembrin, alpinetin, and 5-hydroxy-7-methoxy-flavanone ^4-6. Chalcone and flavanone derivatives were suggested to play an important role as antioxidant agents. Bioassay-guided isolation of the active compounds from CH₂Cl₂: MeOH 1:1 of C. rotunda extract lead to the isolation of panduratin A,4-hydroxy-panduratin A, 5-hydroxy-7-methoxy-flavanone, 5, 7- dihydroxyflavanone, 2′, 6′-dihydroxy-4′-methoxychalcone, and 2′, 4′-dihydro-xy-6′-methoxychalcone. Among them, panduratin A and 4-hydroxypanduratin A might also contribute on antioxidant activity ⁷.

Studies on isolation and bioactivity-tested of plant-origin antioxidants were significantly increased in recent years. To the best of our knowledge, there is very little information available regarding the antioxidant activity of C.rotunda rhizome. We were thus encouraged to investigate and evaluate antioxidant activity of Indonesian C. rotunda rhizome. Antioxidant activity was carried out using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The DPPH test is general bioassays for pharmacological activities of active phytochemical compounds derived from medicinal plants. It is a well-established assay for the in-vitro examination of antioxidant activity in medicinal plant extracts ⁸.

MATERIAL AND METHODS:

Plant Materials: The rhizome of C. rotunda was purchased from Tropical Biopharmaca Research Center, Bogor Agricultural University, West Java, Indonesia.

General Experimental Procedures: Vacuum liquid chromatography (VLC) was performed using Si 60 G (Merck) for column packed and Si 60 (0.2-0.5 mm) (Merck) for sample adsorbed. Column chromatography (CC) was performed using Si 60 (70-230 mesh) (Merck). For TLC analysis, pre-coated silica gel plates (Merck Si 60 GF₂₅₄, 0.25 mm thickness) and Ce(SO₄)₂.4H₂O 1.5% in H₂SO₄ 2N as apparition stain reagent were used. LC-MS analysis was performed using LC-MSXevo G2-S QTOFwith time of flight (TOF) as mass analyses on low power at 4 V, and high power at 25-70 V in duration time 23 min and Quadrupole-time of flight (Q-TOF) was used for mass analysis. The LC system consisted of a C18 column (particle size 2.1 m × 15.0 cm × 1.8 µm) equipped with a guard column containing the same material. The LC was run under gradient system on 0-2 min to 95% water and 5% methanol, isocratic on 2-3 min to 75% water and 25% methanol, gradient system on 14-15 min with 100% water, and isocratic on 19-23 min with 95% water and 5% methanol with 0.2 mL/min flow rate. Chromatogram obtained was analyzed by Masslynx V4.1 software, and mass spectrum was analyzed by ChemSpider, Massbank, and Human Metabolome Database (HMDB) software.

Extraction, Fractionation, and Purification: Dried rhizome of C. rotunda (1.2 kg) was ground and macerated three times with acetone at room temperature gained 161.02 g of crude extract. The crude extract (30 g) then was separated using VLC using various components of n-hexane: EtOAc yielded 8 major fractions (Fr. A-H). Fr. F (466.4 mg) was further fractionated using CC with n-hexane: EtOAc (9:1 to 1:9) resulted in 8 subfractions (Fr. F1-F8). Fr. F2 exhibited 1 spot on TLC using n-hexane: EtOAc 7:3 with R_f value of 0.71 while Fr. F3 showed 2 spots with R_f values of 0.71 and 0.53, respectively on the same eluent. Both fractions then was analyzed using LC-MS.

Antioxidant Activity Assay: Antioxidant activity was examined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ascorbic acid as a positive control. The correlation between concentration and activity was determined, and IC₅₀ was measured using interpolation. Antioxidant activity appears in the value of IC₅₀.

RESULTS AND DISCUSSION: Chromatogram of both Fr. F2 and F3 showed many peaks in range retention time of 0-22 min. Fr. F2 showed 1 peak with high abundance at a retention time of 13.30 minute, while Fr. F3 exhibited 2 peaks at retention time of 11.30 and 13.39 min Fig. 1.

Based on Masslynx software, the peak with a retention time of 13.30 min (Fr.F2) showed similarity with a peak at a retention time of 13.39 min (Fr 3) indicated a compound with the C₁₆H₁₅O₄ molecular formula. The compound had a 99.66% resemblance to the [M+H]⁺271.0975 m/z Fig. 2. According to HMDB, Massbank, and ChemSpider databases, these compounds were classified as chalcone and flavonoids, i.e. pinostrobin (1), cardamonine (2), eqinatine (3), (2E)-1-(4-hydroxy-2- methoxyphenyl)- 3-(4-hydroxyphenyl)- prop- 2-ene-1-one (4), and 7-hydroxy-8-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyrane-4-one (5) Fig. 3.

Compounds 1 and 2 had been isolated from CHCl₃ extract of C. rotunda cultivated in Bangkok, Thailand ⁴. Also, compound 1 was also reported from CH₂Cl₂ extract of C. rotunda cultivated in Pahang, Malaysia ⁹, while compound 2 was also isolated from the methanol extract of C. Rotunda cultivated in other Thailand regions ¹⁰. However, compounds 1 and 2 had not yet reported from other Curcuma species, and compounds 3, 4, and 5 had not yet been reported both in C. rotunda and other Curcuma species.

FIG. 1: THE Fr. F2 (A) AND F3 (B) FRACTIONS CHROMATOGRAM ON LC-MS OF INDONESIAN C. ROTUNDA

FIG. 2: THE MS SPECTRUM FROM 13.30 min (Fr. F2) AND 13.39 min (Fr. F3) RETENTION TIME (A) BASIC SPECTRUM (B) MS FRAGMENTATION SPECTRUM

FIG. 3: PROSPECTIVE COMPOUNDS WITH 13.30 min (Fr. F2) AND 13.39 min (Fr. F3) RETENTION TIME BASED ON HMDB, MASSBANK, AND CHEMSPIDER DATABASE

Fig. 2 showed the [M+H]⁺ of 271.0975 m/z Fig. 2(a) and fragmented peak of 179.0346, 167.0350, 131.0499, and 103.0550 m/z Fig, 2(b). The fragmented peak of a compound corresponding to the values of 167.0350, 131.0499, and 103.0550 m/z was thought to originate from the release of radical ions [C₈H₇], [C₇H₈O₃], and [C₈H₇O₄], respectively. The presumptive compounds according to the fragmentation pattern were compounds 1 and 2 Fig. 3 with fragmentation patterns showed in Fig. 4.

FIG. 4: FRAGMENTATION PATTERN OF COMPOUND 1

According to Masslynx software, the peak with a retention time of 11.30 min (Fr.F3, Fig. 1b) could have C₁₅H₁₃O₄ molecular formula that 99.88% resemblance with [M+H]⁺ of 257.0821 m/z Fig. 5. Based on HMDB, Massbank, and ChemSpider database, the predicted compounds were pinocembrin (6) and liquiritigenin (7) as flavonoid derivatives, chalcone pinocembrin (8) and isoliquiritigenin (9) as chalcone derivatives, and dihydrodaidzein (10) as isoflavonoid derivative Fig. 6. Compound 6 had been reported in CHCl₃: methanol 9:1 extract of C. ecalcarata cultivated in India ¹¹, EtOAc extract of C. rotunda cultivated in Surabaya, Indonesia ¹², and CHCl₃ extract of C. rotunda cultivated in Bangkok, Thailand ⁴. Compound 8 had been reported in the methanol extract of C. rotunda cultivated in Kuala Lumpur, Malaysia ¹³. Compounds 6 and 8 had also been reported on diethyl ether extract of C. rotunda cultivated in Thailand ¹⁴. However, compound 8 had not been reported yet in other Curcuma species and compounds 7, 9, and 10 had also not been reported yet in both C. rotunda and other Curcuma species.

FIG. 5: THE MS SPECTRUM FROM 11.30 min RETENTION TIME (Fr. F3) (A) BASIC SPECTRUM (B) MS FRAGMENTATION SPECTRUM

Fig. 5 showed the values of [M+H]⁺ of 257.0821 m/z Fig. 5(A) and fragmented peak of 179.0343, 167.0346, 153.0191, 131.0499, and 103.0549 m/z Fig. 5(B). The fragmented peak of a compound corresponding to the values of 179.0343, 153.0191, 131.0499, and 103.0549 m/z were thought to arise from the release of successive radical ions [C₆H₅], [C₈H₇], [C₆H₅O₃], and [C₇H₅O₄]. The alleged compounds according to the fragmentation pattern were compounds 6 and 8. Fig. 7 showed fragment ions from compound 6.

FIG. 6: PROSPECTIVE COMPOUNDS WITH 11.30 min RETENTION TIME (Fr. F3) BASED ON HMDB, MASS BANK, AND CHEMSPIDER DATABASE

The result scavenging effect to the DPPH free radical of the acetone extract of Indonesian C. rotunda rhizome, ascorbic acid, Fr. F2, and F3 showed that none of the fractions was found to be more active than ascorbic acid (IC₅₀ = 4.62 ppm). The acetone extract exhibited a moderate antioxidant activity with IC₅₀ value of 125.75 ppm. However, this value was greater than Pratiwi ¹⁶ which reported the IC₅₀ value of 100.24 μg/mL. Fr. F2 and F3 could not be determined for their antioxidant activity because their inhibition value was less than 50%.

FIG. 7: FRAGMENTATION PATTERN OF COMPOUND 6

According to Tanjung et al., ¹² compounds 2 showed the IC₅₀ value of 21.54 μg/mL against DPPH. The difference was presumably due to several factors, such as plant genetic factors, growing areas, environmental stresses such as heavy metals or ultraviolet exposure, and physical factors such as temperature, humidity, and climate ¹⁶. Phenolic and flavonoid in all accessions of C. rotunda were not responsible for their antioxidant activity, or alternatively, that is radical scavenging activity was due to specific phenol or flavonoid compounds. In other medicinal plants, the results of that study are in agreement with other reports ¹⁷.

CONCLUSION: The secondary metabolites had been isolated from acetone extract of Indonesian C. rotunda rhizomes. Separation and identification used various chromatography methods and LC-MS, respectively, showed a dominant peak on the retention time of 13.30 and 11.30 min suspected pinostrobin (chalcone) and pinocembrin (flavanone), respectively. Antioxidant activity towards DPPH of crude extract showed moderate activity with IC₅₀ of 125.75 ppm. However, the antioxidant activities from both isolated fractions could not be determined due to less than 50% of its inhibition percentage.

ACKNOWLEDGEMENT: The authors are grateful to Department of Chemistry, Faculty of Mathematics and Sciences, Bogor Agriculture University for financial support. We also thank to Department of Chemistry, the Faculty of Mathematics and Sciences, Bandung Institute of Technology for providing laboratory facilities, specially 1D and 2D NMR.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Rahayu DUC, Nurfadhilah D and Sugita P: Phytochemical study of Indonesian Curcuma rotunda rhizome and its antioxidant activity towards 1,1-diphenyl-2-picrylhydrazyl (DPPH). Int J Pharm Sci & Res 2019; 10(7): 3354-58. doi: 10.13040/IJPSR.0975-8232.10(7).3354-58.

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