Mechanical Extraction and Antioxidant Activity of Gambier (Uncaria gambier Roxb) Leaves and Twigs

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The Mechanically Extraction Process from Leaves and Twigs of Gambier (Uncaria gambier Roxb) and Its Antioxidant Activity

Galuh Widiyarti^*, Andini Sundowo, Euis Filailla and Joddy Arya Laksmono

Research Center for Chemistry, Indonesian Institute of Sciences (LIPI), PUSPIPTEK Serpong, Tangerang Selatan, Banten 15314, Indonesia

*Corresponding email : [email protected] ; [email protected]

Received 13 November 2019; Accepted 15 March 2020

ABSTRACT

The extraction process from leaves and twigs of gambier (Uncaria gambier Roxb) plant was conducted mechanically by using traditional hydraulic press, conventional screw press, and modified twin-screw press. The leaves and twigs of gambier plant was obtained from traditional farmer in Limapuluh Kota, West Sumatera, Indonesia. The water, ash and catechin contents of the gambier extracts were analyzed based on SNI 01-3391-2000 using spectrophotometry and thermogravimetry method. Antioxidant activity analysis of the extracts was also performed by 1,1-diphenyl-2-picrylhidrazyl (DPPH) method and compared to vitamin C as a standard antioxidant. The analysis results showed that the extracts contain no ash. Meanwhile, the catechin and water contents of the extracts were approximately 50 and 13% thus the extracts were classified as quality gambier 2. Other than that, analysis result of catechin and epicatechin contents of the extracts using HPLC and compared to the reference materials showed that catechins contents of the extracts using traditional hydraulic press, conventional screw press, and modified twin-screw press give catechin content in about 94.296-95.030%. However, epicatechin was detected in a trace amount. The antioxidant activity of the extracts were 2.5 times stronger than reference. The IC50 value of 4.37-4.52 µg/mL and was categorized as active antioxidant.

Keywords: Gambier, mechanically extraction, antioxidant, DPPH

INTRODUCTION

Gambier (Uncaria gambier Roxb) plants belong to the genus Uncaria and the family of Rubiaceae [1]. Uncaria is often used as traditional medicine, such as a drug for burns, digestive disorders, fevers, headaches, and as an antibacterial or antifungal [2]. Gambier plants have many benefits for health, such as antioxidants, antihiperlipidemia, and antibacterial [3]. Gambier extract can be prepared from the leaves and twigs part. The process involves of the removing of the sap and then boilling. The other process use aqueous extraction, by squeezing, drying, and molding in various forms [4]. In Indonesia, gambier is traditionally used as a mixture of chewing. Other than that, gambier is also used as a medicine for diarrhea, dysentery, mouthwashs, mouth ulcers, mixed ingredients of cosmetics, plywood adhesives, tanners, and natural dyes [5-8].

Gambier also become an important agricultural commodities for export of Indonesia.

Several major destination countries such as Bangladesh, India, Pakistan, Taiwan, Japan, South Korea, France, and Switzerland [9]. Almost 80% of global’s gambier supplied are

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originated from the Province of West Sumatera, especially from the District of Limapuluh Kota.

The important of chemical composition of gambier mainly composed of flavonoid class, such as catechin and epicatechin. The other component is alkaloids [7]. The quality parameter in the market mainly also based on the catechin content. The minimum catechin content of gambier classified into 3 levels or class. Level I is gambier with cathechin content above 60%, level II for gambier with catechin content about 50%, and level III for gambier with catechin content below 40% [4]. This cathecin in gambier very important due to its activity as antioxidant and antidiabetic [10-13], antibacterial against gram positive bacteria such as Staphyloccoccus aureus, Bacillus subtilis, and Streptoccus mutans [14,15], anti hypercholesterolimic [16], and anticancer [17-19].

The problem of gambier production, particularly in Limapuluh Kota is related to the quality and hygiene of the gambier products. The research on the application of appropriate technology for gambier production aims to improve the yield of gambier production and maintain the quality of gambier products. In this research, the extraction process of gambier was done mechanically using a modified twin-screw press system. The extraction process was expected to produce gambier extract with better yield and quality comparing to other mechanically extraction methods. This research was conducted in 3 steps, the first step was the pre-tretment of gambir leaves and twigs by cleaning, washing and choping of gambier leaves and twigs, the second step of aqueous extraction process of gambier leaves and twigs, and the third step of the physical chemical analysis of gambier extract products. The quality of gambier extracts was performed by calculating the water and ash content. And also, catechin contents of the extracts according to Indonesian National Standard (SNI) number 01- 3391-2000 for gambier [4]. The catechin, include epicatechin contents of the extracts were analyzed using high performance liquid chromatography (HPLC) and compared to the reference materials [20]. Meanwhile, the antioxidant activity of the extracts were tested as radical scavenger activity by using DPPH method [21,22].

EXPERIMENT

Chemicals and Instrumentation

The materials used in this study were leaves and twigs of gambier plants obtained from traditional farmer in Limapuluh Kota district, West Sumatra of Indonesia. Chemicals used i.e.

1,1-diphenyl-2-picrylhidrazyl (DPPH) (TCI D4313), vitamin C (ascorbic acid) (EM 100468) for antioxidant test, catechin (Sigma-Aldrigh C1251), epicatechin (Sigma-Aldrich E1753) and methanol.

The equipments used for research includes traditional-hydraulic press, conventional hydraulic press, and modified twin-screw press for extraction process of leaves and twigs from gambier plant. High performance liquid chromatograph (Agilent Technology 1260 Infinity II, C18 column, eluent acetonitrile/water/phosphate buffer with 5/45/50 in volume ratio, phosphate buffer pH 2.5, 20 mM, flow rate 0.8 mL/min, using DAD in UV wavelength detection) for catechin analysis. UV-Vis spectrophotometer (Agilent Cary 60) was operated for antioxidant activity analysis of gambier extracts, oven drying for analysis water content and furnace for analysis ash content of gambier extracts.

Procedure of extraction

The extraction of leaves and twigs from gambier plant was conducted using three type equipments. They were traditionally hydraulic press, conventional screw press, and modified twin-screw press. A dried leaves and twigs (10 Kg) was boiled using 100 L water to soften

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the materials for about hours. Hot leaves and twigs as raw materials were then putted in to the container which is part of each of the extraction units. The raw materials were extracted using traditionally hydraulic press, conventional screw press, and modified twin-screw press, respectively.The liquid extract was then dried using drying oven until a crude gambier extracts were obtained. The yield of extraction process was calculated as dry basis of extract weight compared to the sample weight.

Water, ash and catechin analysis

Procedure to analysis water and ash content of gambier following reference cited in National Standard of Indonesia (SNI) number 01-3391-2000 [4] for food and bavarage analysis. The gambier sample was heated in oven (105 ^oC). It was further weight until a contants weight was achieved. The water content is calculated by dividing with initial weight of sample. Meanwhile, The ash content was determined from the dried sample of gambir (3.0 g) in crucible porcelain. It was heated in furnace at 550 ^oC. The furnace was turn off and temperature was dropped to room temperature. The sample was put in desicator, and weigh until constant weight was achieved.

Catechin was analyzed twice, firt procedure according to the SNI 01-3391-2000 [4].

Series of catechin standard were prepared with variation in concentration. Then each was analyzed using UV-Vis spectrophotometer. Plott standard of catechin was created between absorbance and concentration. Meanwhile, gambier extract was also analyzed similarly, and the absorbance value was plotted the standard curve to provide concentration of catechin content in gambier extract.

The second procedure for catechin analysis was performed by using high perfomance liquid chromatograph (HPLC). This procedure followed Fernandez et al. [20] and similar paper was reported [23]. Qualitative analysis of catechin was determined using similarity of retention the catechin in the sample toward the standard. Meanwhile, the quantitaive analysis was based on the area percentage calculated from peak chromatogram of catechin. The detailed procedure as following. Gambier sample (0.5 g) was extracted with acetonitrile/water at room temperature for an hour. The extract was filterred off and dilluted to 100 mL in callibrated flask. The aliquot of the sample was diluted and injected (5.0 µL) in the HPLC. The chromatogram of gambier extracts resulted was compared to the chromatogram of the standard catechin (Sigma-Aldrigh C1251) and epicatechin (Sigma- Aldrich E1753) as the reference materials [20].

Sample analysis was undertaken to the gambier extract obtained from traditional hydraulic press, conventional screw press, and modified twin-screw press techniques. Include the sample of reference for standard catechin/epicatechin.

Antioxidant activity analysis

The gambier extracts were analyzed its antioxidant activity by measuring radical scavenging capability of the extract using DPPH method. The procedure was undertaken according to reference [21]. The antioxidant activity of the gambier extracts compared to the vitamin C (ascorbic acid) as a positive control. Besides that, methanol was used as a negative control.

A 2.0 mg of vitamin C was dissolved in methanol (2.0 mL). After that, a series solution of 2.5-20 µg/ml of the standard solution were prepared. Meanwhile, the sample solutions were prepared by dissolving of 2.0 mg of gambier extract in 2.0 mL of methanol. Then, a series solution of samples of 5-50 µg/mL were prepared. The negative control and sample solutions were added with 500 µL of DPPH solution and incubated for 30 minutes in the dark

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place. After that, each sample was measured their absorbance using UV-Vis spectrophotometer at 515 nm. The value of % inhibition was calculated, and then the IC50

value as the concentration that caused 50% inhibition of the radical from DPPH was calculated [21,22].

RESULT AND DISCUSSION Gambier extracts yield

Three method of extractions were compared, i.e traditional hydraulic press, conventional screw press, and modified twin-screw press. The yield of the gambier extracts was calculated based on the percent weight of the extracted gambier divided by the weight of sample from the leaves and twigs. The yield resulted is summarized in Table 1, and the result give slightly different quantity. The highest yield of gambier extract was obtained by using modified twin-screw press technique. The gambier extract was resulted in 20% yield. This technique undergoes mechanically, and allow the raw sample have extensive residence time in the counter-current contact with twin-screw press surface. The longer of this contact produce intensively the aqueous extract of gambier [24]. The second higher yield was given from conventional screw press technique. A 14% yield of gambier extract was collected. This method undergoes in one-way of screw pressing process, that not extesively allow the sample contact with the surface of screw press compare to the twin-screw press model. And lastly, the lowest yield (9.0%) of gambier extract was proccessed under traditional hydraulic press.

This method provide a limited surface area interaction between the sample and the piston surface of the instrument. Thus, mechanical force resulted occurs at one-side surface, and this give a modest pressure to the sample. As a result, the quantity of the gambier extracted is low.

Table 1. Yields of gambier extraction process.

Entry Extraction process Yield (%) 1 Traditional hydraulic press 9.0 2 Conventional screw press 14 3 Modified twin-screw press 20

Table 2. Water, ash, and catechin content of gambier extracts.

Entry Extraction process Water (%)

Ash (%)

Catechin (%)^* 1 Traditional hydraulic press 13.81 0 52.25 2 Conventional screw press 13.81 0 52.71 3 Modified twin screw press 13.21 0 56.07

*Catechin analysis was undertaken following SNI 01-3391-2000

The water and ash content in gambier extract

The water and ash content composed of the gambier extract which are resulted from traditional hydraulic press, conventional screw press, and modified twin-screw press are tabulted in Table 2. In overal, no different water content was recorded and no ash content was detected. Water content was measured by heating of the gambier extract in the oven at 105 ^oC following the Indonesian National Standar (SNI) for Gambier with SNI number 01-3391-

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2000 [4]. Including the ash content, the gambier extract was heated in the furnace in about 550 ^oC. Moreover, the catechin content from each extract was detected in between 52.25%

and 56.07%. The highest of catechin content of the gambier extract was given from the extraction technique using a modified twin-screw press. According to the SNI, these extract was categorized as Class II of Gambier product.

Catechin in gambier extract

Catechin are basically have four different structure, especially their stereochemical orientation. The epimer of catechin was named epicatechin. The catechin itself have two stereoisomer structure, i.e. (+)-(2R,3S)-catechin and (-)-(2S,3R)-catechin. Meanwhile, catechin epimer or epicatechin also have two stereoisomer, i.e. (+)-(2S,3S)-epicatechin and (-)-(2R,3R)-epicatechin (Figure 2). All of them are known as condensed tannin.

OH OH

HO O

OH OH

HO O

OH OH

HO O

OH OH

HO O

OH OH

(+)-(2R,3S)-catechin (-)-(2S,3R)-catechin (-)-(2R,3R)-epicatechin (+)-(2S,3S)-epicatechin

Figure 2. Chemical structure of catechin and epicatechin

Catechin analysis from gambier extracts following SNI 01-3391-2000 in all techniques extraction give composition in 52.25-56.07% (Table 2). This result basically is combination from all of the stereochemical structures both from catechin and epicatechin. Previous study conducted by Rahman et al. [25] using local gambier and collected from Solok Bio-Bio Limapuluh Kota district gave similar yield. Further purification of the result using hot water (70 ^oC) under stirring for an hour, then soxhlet extraction with ethyl acetate gave gambier extract with a high content of catechin. It was in 97.40% purity. Before this report, Widiyarti et al. [26] also published the purification strategy of gambier extract. The sample were taken from local market, and purification was undertaken with maceration and soxhletation techniques. Ethyl acetate was used as a solvent. The result showed maceration could provide gambier extract in 41.8% yield meanwhile soxhletation provided 41.4% yield. The catechin content for both techniques were detected in 91.97 and 97.99% purity.

Table 3. Catechin content of gambier extracts analyzed with HPLC Entry Extraction process Retention time

(min.) Catechin (%) 1 Traditional hydraulic press 7.693 94.509 2 Conventional screw press 7.701 94.296 3 Modified twin screw press 7.696 95.030

Meanwhile, in this research, catechin analysis from the extracted gambier were undertaken using HPLC. The chromatograms resulted were displayed in Figure 3, and the summary as in Table 3. The catechin and epicatechin standard was analyzed for comparison. Catechin came up in the retention time about 7.70 minute and epicatechin in 14.87 minutes. The extracted gambier from the traditional hydraulic press, conventional screw press, and modified twin- screw press indicate a major catechin content in similar retention time to the standard. Their

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percentage in the extract about 94.296-94.509 %, meanwhile the epicatechin was detected as a trace.

Figure 2. HPLC chromatogram of the gambier extract produced by traditional hydraulic press (A), conventional screw press (B), and modified twin-screw press (C). The peak of chromatogram symbolized as (C) is associated to the peak of catechin and (EC) for epicatechin to the reference sample.

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Antioxidant activity of the gambier extracts

The result from antioxidant activity evaluation of the extracted gambier was depicted in Figure 3. The data is presented as IC50 value of each sample and vitamic C as a reference.

Figure 3. The IC50 value of antioxidant activity of the gambier extracts produced by traditional hydraulic press(G1), conventional hydraulic press (G2), modified twin screw press (G3), and vitamin C as reference. The value with 5% of error deviation.

The IC50 for each gambier extract sample give a better capability to inhibit a free radical compare to the vitamin C as a reference. Their capabilities almost two and half time stronger. The gambier extract has IC50 about 4.37-4.52 µg/mL, meanwhile the reference has IC50 10.97 µg/mL. According to Yen et al. [21], the sample of gambier extract was categorized as very active or strong antioxidant. The IC50 value about 50–100 µg/mL was categorized as strong antioxidant. The IC50 value about 100–150 µg/mL was categorized as active/moderate antioxidant, and weak antioxidant if the IC50 value about 151–200 µg/mL [21]. Previous result reported by Widiyarti et al. [13] indicated slightly similar finding. After purification by maceration and soxhletation using ethyl acetate as solvent could give the IC50

about 4.55-18.23 µg/mL [13].

CONCLUSION

The extraction process of gambier from leaves and twigs of gambier plants using traditional hydraulic press, conventional screw press, and modified twin-screw press resulted gambier extract yield in about 9.0-20%. The catechin content still in between 52.25% and 56.07% following SNI 01-3391-2000 procedure. Meawhile HPLC analysis can detect in 94.296-95.030%. Further antioxidant analysis also give IC50 value in narrow range (4.37-4.52 µg/mL).

CONFLICT OF INTEREST

Author declare that there is no conflict of interest ACKNOWLEDGMENT

The authors would like to thank Indonesian Ministry for Research Technology and Higher Education (Ristekdikti) for funding this research through a Incentive for Enhancing

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Capability Researchers and Engineers Program FY 2012 and the individual National Innovation System Research Incentive (INSINAS) Program FY 2019.

REFERENCES

[1] Heitzman, M.E., Neto, C.C., Winiarz, E., Vaisberg, A.J., and Hammond, G.B., Phytochem., 2005, 66 (1), 5-29.

[2] Vellury, R., Weir, T.L., Bais, H.P., Stermizt, F.R., and Vivanco, G.M., J. Agric. Food Chem., 2004, 52 (5), 1077-1082.

[3] Alegantina, S., and Setyorini, H.A., Jurnal Kefarmasian Indonesia, 2017, 7 (1), 10-18.

[4] Indonesian National Standard (Standar Nasional Indonesia) Gambir: SNI 01-3391- 2000. Jakarta: Badan Standarisasi Nasional, 2000, 1-6.

[5] Lucida, H., Bakhtiar, A., and Putri, W.A., J. Sains Tek. Far., 2007, 12 ((1), 1-7.

[6] Rahmawati, N., Bakhtiar, A. and Putra, D.P., Jurnal Penelitian Farmasi Indonesia, 2012, 1 (1), 1-6.

[7] Kasim, A., Asben, A., and Muhtiar, S., Majalah Kulit, Karet dan Plastik, 2015, 31 (1), 55-64.

[8] Sofyan, S. and Failisnur, F., Jurnal Litbang Industri, 2016, 6(2), 89-98.

[9] Gumbira-Sa’id, E.K., Syamsu, E.M., AH, B. And NA, E., Asia Forum on Business Education (AFBE) Journal, 2010, 3(1), 145-160.

[10] Arakawa, H., Maeda, M., Okubo, S. And Shimamura, T., Biol. Pharm. Bull., 2004, 27 (3), 277-288.

[11] Rauf, R., Santoso U., and Suparmo, S., Agritech, 2010, 30 (1), 1-5.

[12] Ahmad, R., Hashim, H.M., Noor, Z.M., Ismail, N.H., Salim, F., Lajis, N.H. and Shaari, K., Res. J. Med. Plant, 2011, 5 (5), 587-595.

[13] Widiyarti, G. and Sundowo, A., Jurnal Ilmu Kefarmasian Indonesia, 2012, 10(2), 81- 86.

[14] Pambayun, R., Gardjito, M,. Sudarmadji, S. and Kuswanto, K.R., Majalah Farmasi Indonesia, 2007, 18(3), 141-146.

[15] Widiyarti, G., Sundowo, A. and Angelina, M., Jurnal Ilmu Kefarmasian Indonesia, 2014, 12(2), 145-153.

[16] Frinanda, D., Efrizal, E. And Rahayu, R., Jurnal Biologi UNAND, 2014, 3(3), 231-237.

[17] Pilarski, R., Filip, B., Wietrzyk, J., Kuras, M. and Gulewicz, K., Phytomedicine, 2010, 17(14), 1133-1137.

[18] Widiyarti, G., Sundowo, A., and Ernawati, T., Proceeding International Seminar on Translational Research in Cancer Chemoprevention, 2012, 57-62.

[19] Caon, T., Kaiser, S., Feltrin, C., de Carvalho, A., Sincero, C.T.M., Ortega, G.G. and Simoes, C.M.O., Food Chem. Toxicol., 2014, 66, 30-35.

[20] Fernandez, P.L., Martin, M.J., Gonzales, A.G. and Pablos, F., Analyst, 2000, 125(3), 421-425.

[21] Yen, G.C. and Chen, H.Y., J. Agric. Food Chem., 1995, 43(1), 27-32.

[22] Shekhar, T.C. and Anju, G., Am. J. Ethnomed., 2014, 1(4), 244-249.

[23] Sudjatini, S., Agrotech., 2017, 2 (1), 43-49.

[24] Evon, P.H., Kartika, I.A., Cerny, M. and Rigal, L., Ind. Crops Prod., 2013, 47, 33-42.

[25] Rahman, E.D., Sari, E., Burmawi, B., Frizka, F. and Endah, E., in IOP Conf. Series:

Materials Science and Engineering, 2018, 316(1), 012022.

[26] Widiyarti, G., Sundowo, A., and Hanafi, M., Makara J. Sci., 2012, 15(2), 129-134.