TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITIES IN CALLUS OF KAEMPFERIA PARVIFLORA

Black ginger or Kaempferia parvi lora is one useful medicinal plant that urges the need to be cultivated widely. This plant has some previous studies about their rhizomes and callus growth optimization, but their total phenolic content and antioxidant activities are not very well known. This study aims to study the total phenolic content and antioxidant activities of callus of K.parvi lora. This study is to determine the type and concentration of hormone that give the best effect on the growth of callus in vitro culture from different parts (meristem, andbasal part) of K.parvi lora. Different concentrationswere tested withmedium2,4-dichlorophenoxyacetic acid (2,4-D) andnapthyleneacetic acid (NAA) in callus cultivation. The highest total phenolic content tested in callus is one with 10.00 mg/L 2,4-D from the meristem part of the plant. The highest antioxidant activity is from themeristempart of the plantwith 5.00mg/L 2,4-D.


INTRODUCTION
The ginger plant Kaempferia parvi lora belongs to the Zingiberaceae family. It is also known as "kunyit hitam" in Malay, and Krachai-dam in the Thai language. This plant has long been utilized in Malay and Thai medicine to promote health, relieve body pains, treat leucorrhea, mouth disease, gastrointestinal issues, and correct male impotence Tewtrakul et al. (2008a), Rujjanawate et al. (2005). K. parvi lora has been proven by studies to have anti-allergic Tewtrakul et al. (2008b), anti-fungal Yenjai et al. (2004), anti-ulcer Rujjanawate et al. (2005), and antimicrobial properties Yenjai et al. (2004). Wide medicinal usage of K. parvi lora made this plant have high needs to be cultivated widely. The issue of having a low supply of high-quality stock triggered the effort to study the production of high-quality K. parvi lora. There are studies of producing K. parvi lora by in vitro culture Prathanturarug et al. (2000). The availability of superior seeds in large quantities and pathogens free depends on the type and concentration of hormone added to the media Sumadi (2020). Phenolic compound plays a vital role in having anti-microbial and anti-fungal properties as they have these redox properties, which plays an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxide Ghasemzadeh et al. (2010). Many authors suggested that total phenolic content has a direct link to antioxidant activities because of phenolic compounds, due to their hydroxyl groups which have a high scavenging capacity to combat free radicals.
This study aims to determine the best concentration of mediums that give a positive effect on callus from different parts of K. parvi lora (meristem and basal part). In plants, the meristem is an area of cells capable of division and growth. They are classi ied by their location in the plant as apical (located at the root and shoot tips), lateral (in the vascular and cork cambia), and intercalary (at internodes, or stem regions between the places at which leaves attach or leaf bases) Britannica (2020). While the basal part is de ined as the lowest or bottom part of an object on which it stands or the main part to which other parts are added Baluška et al. (2005). Mediums used contain two types of auxin hormone which are 2,4-dichlorophenoxyacetic acid (2,4-D) and napthyleneacetic acid (NAA) with different concentrations (0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 3.0 mg/L, 5.0 mg/L and 10 mg/L).

INITIAL CULTURES ESTABLISHMENT
The cultivation of Kaempferia parvi lora took place inside a glasshouse located at the Malaysian Agriculture Research and Development Institute (MARDI), Serdang. The shoots of the plants were then collected and act as explants. The shoots were cleaned under running tap water for an hour. The cleaning process was followed by commercial laboratory detergent and rinsed with water. water. The explants were submerged in 1 % (v/v) of fungicide (Benomyl 50 %, Benlate ® ) for an hour and then proceed with rinsing of tap water. Subsequently, Clorox was used as part of the sterilization process and followed by a few rounds of cleaning and rinsing with sterile distilled water. A sterile surgical blade was used to cut the leaf sheath present on the buds of Kaempferia parvi lora. The explants were inoculated onto basal Murashige and Skoog's (1962) medium with 3.0 mg/L of benzyl aminopurine (BAP) for the production of plantlets of Kaempferia parvi lora. The pH was adjusted to 5.8 and eventually followed by autoclaving at 121°C under 1.05 kg/cm 2 for 15 minutes. The cultures were grown under white luorescent light (3,000 lux) with a photoperiod duration of 16 hours light and 8 hours darkness at 25 ± 2°C. The plantlets cultured in vitro were used to induce callus.

CALLUS INDUCTION
Callus induction of Kaempferia parvi lora initially started with plantlets that were grown in vitro and two parts of the explants which are the meristem and basal part were sub-cultured separately inside a 150 ml lask with a liquid medium consisting of MS medium supplemented with 60 % sucrose for callus induction with two treatments which are 2,4-D and NAA with a variety of concentration ( 0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 3.0 mg/L, 5.0 mg/L, 10.0 mg/L). The plantlets were sub-cultured every month interval for three months. After three months, the callus was dried at a temperature of 40°C -50°C for three days.

DETERMINATION OF PHENOLIC CONTENT
The total phenolic content (TPC) of the dried callus was determined using the Folin-Ciocalteu (FC) method. Approximately 0.1g of dry callus were added to 0.2 ml of FC reagent (5-fold diluted with distilled water). The mixture was shaken for 3 minutes and then sodium carbonate with a volume of 0.2 ml (10 % w/v) was added. Under the dark condition, the mixture was left to stand for 30 minutes. The absorbance was read using Jasco V-550 UV-VIS spectrophotometer at 760 nm. TPC was expressed as milligram gallic acid equivalent per gram dry extract (mg GAE/mg dry extract).

DETERMINATION OF ANTIOXIDANT ACTIVITY
DPPH radical scavenging assay was used to determine the antioxidant activity of the basal part and the meristem of the callus. 0.5 ml of callus extract along with 4.5 ml of DPPH were put inside 250 ml lask and methanol was added. Subsequently, the mixture was shaken and left to stand at room temperature for 30 minutes under dark conditions. The antioxidant activity was determined at 517 nm against blank solvent. The antioxidant activity was recorded for different treatments with different concentrations for two different parts which are the meristem and basal part.

RESULT AND DISCUSSION
Based on a previous study, both culture media (2,4-D and NAA) are suitable for callus induction using shoot buds explants Zuraida et al. (2014). However, the matter of their phenolic content has not yet been discovered. Therefore, the purpose of this study is to identify the optimal concentration of media that produce the highest phenolic content and also antioxidant activities for both basal and meristem part of K. parvi lora.
As shown in Figure 1 , the meristem of K. parvi lora cultured on 2,4-D media with the concentration of 10.0 mg/L has the highest accumulation of phenolic compound (1340 U/g dry sample), while the basal part is at its highest accumulation with 5.0 mg/L of 2,4-D (653 U/g dry samples). On the other hand, the highest phenolic accumulation in NAA for meristem and basal part is at 10.0 mg/L where meristem recorded for 1198 U/g dry sample and basal part recorded for only 489 U/g dry sam-ples. Generally, callus cultured in NAA have lower phenolic content when compared with callus cultures in 2,4-D. However, in both meristem and basal parts at 1.0 mg/L, NAA relatively has higher reading compared to 2,4-D with 942 U/g dry samples in 1.0 mg/L of NAA compared to only 893 U/g dry samples in 1.0 mg/L of 2,4-D for meristem. A similar pattern can be observed also in 1.0 mg/L where 1.0 mg/L of NAA was recorded for 324 U/g dry samples compared with the readings recorded for 322 U/g dry samples at 1.0 mg/L of 2,4-D for the basal part.
Other than that, meristem parts are seen to produce greater phenolic content compared to the basal part. Phenolic content correlates with the antioxidant properties as phenolic content in the plants or this research is K. parvi lora have redox properties that can act as antioxidant Johari and Khong (2019). According to a study Vichitphan et al. (2007) that was also conducted on K. parvi lora, phenolic content is the most crucial part in determining antioxidant potentials, and also in the same research proved a positive correlation between phenolic content and also antioxidant activity. A positive correlation can also be seen regarding the results obtained in this research whereby for meristem cultured in 5.0 mg/L of 2,4-D showed high antioxidant activity (114 U/g dry samples) with the phenolic content that is also high (1090 U/Gram Dry Sample). However, a positive correlation cannot be seen in 10.0 mg/L of 2,4-D where high phenolic content 1340 U/Gram Dry Sample have low antioxidant activity (76 U/Gram Dry Sample). This phenomenon may be caused by the breaking down of the phenolic compound whereby it oxidized which consequently inhibit the activity of antioxidant. The antioxidant activities of two distinct parts (meristem and basal parts) of K. parvi lora are shown in Figure 2 . Callus from the meristem and basal parts K. parvilora are cultivated in various concentrations of 2,4-D and NAA media. The meristem of K. parvi lora cultured on 2,4-D media with the concentration of 5.0 mg/L has the highest antioxidant activities among all other treatments (114 U/g dry samples) and as for the basal part, the highest reading is only at 39 U/g dry samples for 1.0 mg/L of 2,4-D. This indicates that the basal part does not contain as much antioxidant as the meristem. As for NAA, both meristem and basal parts peaked at 0.5 mg/L with results recorded at 108 U/g dry samples and 46 U/g dry samples respectively. However, in a comparison between both treatments for the basal part, NAA showed higher readings than 2,4-D with the readings recorded at 46 U/g dry samples at 5.0 mg/L. As for meristem, when compared, 5.0 mg/L still showed the highest reading for antioxidant activity with 114 U/g dry samples but in this case, 2,4-D showed higher reading when compared to NAA (108 U/g dry samples).
Based on the results, it can be said that the 5.0 mg/L of 2,4-D is the optimal concentration to induce the highest amount of antioxidant activity in Kaempferia parvi lora and from the results, it can be seen that meristem have better antioxidant content. However, a study PRIHANTINI et al. (2018) stated that NAA is better in callus induction but the research only used 0.5 mg/L of 2,4-D as the highest concentration for 2,4-D. On the other hand, this research proved that a high concentration of 2,4-D produced callus that contains high antioxidant activity and the results seem to be parallel with another study El-Nabarawy et al. (2015) that proved that 1.0 mg/L of 2,4-D but in a combination of 0.5 mg/L BA are suitable for callus formation for ginger. Furthermore, the same study also stated that NAA, BA, or even the combination of both NAA and BA were unable to induce callus. Hence, proving a point that 2,4-D is suitable to produce callus and from the callus induction, antioxidant activity can be identi ied. In this research, meristem can be seen to be better than basal parts in producing higher antioxidant content. Similar indings can be seen in a study Jiménez-Aspee et al. (2018) that stated the meristem part has higher antioxidant activity compared to other parts of plants.
The success of micropropagation is dependent on several parameters, including plant growth regulators and the type of explants used Pati et al. (2006). Plant growth regulators operate as signals in the developmental processes of plants, stimulating, inhibiting, or regulating growth MERCIER et al. (1997). 2,4-D and NAA are types of auxin hormones which are growth hormones that are produced at the apical tips of the growing plants. An equal level of hormones was found to induce the best callus formation Chaâbani et al. (2015).
Our indings imply that a medium containing plant growth regulator activates the phenylpropanoid pathway in which boosts the production of phenolic substances Johri and Mitra (2001). In the phenylpropanoid pathway, the most important enzyme is the phenylalanine ammonia-lyase enzyme which acts as the main biosynthesis of many phenolic compounds in plants such as the synthesis of phenolic, lavonoids, and lignin compounds Schuster and Retey (1995).
Reports are saying that the antioxidant activity has a direct relation with the number of phenolic compounds Krajnc et al. (2013), Dias et al. (2016). Some authors said that in response to biotic and abiotic elicitors, antioxidant activity and phenolic compounds in different plants have a positive association Vasco et al. (2008).

CONCLUSION
High phenolic and antioxidant activity in the callus of K. parvi lora can be obtained by using the meristem part from K. parvi lora. The best medium for a high induction of total phenolic content and antioxidant activity as in callus culture for this plant was 2,4-D with a concentration of 10.0 mg/L and 5.0 mg/L, respectively. The antioxidants in K. parvi lora are vital and crucial for the human health and hence this research provided optimal concentration for callus of K. parvi lora that are able to produce high antioxidant and further prove that meristems have better antioxidants content.