Chemical profile, anti 5-lipoxygenase and cyclooxegenase inhibitory effects of ginger ( Zingiber officinale ) rhizome, callus and callus treated with elicitors

Chemical profile, anti 5-lipoxygenase and cyclooxegenase inhibitory effects of ginger ( Zingiber officinale )... ABSTRACT : The present study investigated the chemical profiles and evaluated the inhibitory effect against 5-Lipoxygenase (5-Lox) activity for extracts of ginger rhizome, callus, and callus treated with the elicitors; yeast extract (100, 300 and 500 mg/L), glycine (100, 200 and 300 mg/L) and salicylic acid (100 and 200 mg/L). Oils and chloroform: methanol (CM) extracts were prepared by maceration in petroleum ether and CM (1:1, v/v), respectively. Chemical profiles were determined by gas chromatography/mass spectrometry (GC/MS) analysis. Oil of the callus recorded higher 5-Lox inhibitory effect (IC 50 58.33±4.66 μg/mL) than the oil of rhizome (IC 50 168.34±15.64 μg/mL) and comparable to that of the positive control; Nordihydroguaiaretic acid (IC 50 61.25±1.02 μg/mL). The chemical profile of the callus oil contained large amounts of fatty acids, mainly the unsaturated fatty acid oleic acid (31.11%) and saturated fatty acid palmitic acid (28.56%). Elicitors modified the chemical profile of the callus and ameliorated the anti-5-Lox activity of CM extract of the callus. CM extracts of callus treated with 100 and 300 mg/L yeast extract and 50 mg/L salicylic acid significantly suppressed (P ≤ 0.05) the 5-Lox activity by 33.16%, 25.46% and 16%, respectively as compared to the CM extract of untreated callus. In conclusion, ginger callus could be considered as a valuable dietary supplement in the treatment of various inflammatory disorders.


INTRODUCTION
Ginger (Zingiber officinale Roscoe) is widely consumed as spice and to cure a wide range of diseases (KARAMAN et al., 2019). Pharmacological studies revealed that ginger has broad healthy benefits such as anti-emetic (KARAMAN et al., Ciência Rural, v.52, n.10, 2022. Ali et al. The healthy properties of ginger are frequently imputed to some pungent or non-volatile components in ginger as gingerols and shogaols or to volatile components as zingiberene, which is mainly responsible of the distinct aroma of ginger (PRASAD & TYAGI, 2015). These phytocompounds exist with varying concentrations depending on ginger form used, either fresh or dry, and method of extraction. Fresh rhizome contains high amount of gingerol which is converted to shogaol form via heating or drying process (PRASAD & TYAGI, 2015). Lipoxygenases are a family of nonheme ironcontaining enzymes that catalyze the deoxygenation of polyunsaturated fatty acids (PUFAs). Various lipoxygenases are involved in the metabolism of leukotrienes, a family of eicosanoid inflammatory mediators. For example, leukotrienes are synthesized in the cell from arachidonic acid by arachidonate 5-lipoxygenase (5-Lox). 5-LOX plays a pivot role in asthma and inflammation, as it causes the constriction of bronchioles in response to cysteinyl leukotrienes such as LTC4, thus leading to asthma. It also induces neutrophilic inflammation by its recruitment in response to LTB4. Ginger extracts as well as gingerol, shogaol, and other structurallyrelated substances in ginger exhibited a broad spectrum of anti-inflammatory activities through multiple mechanisms as suppressing prostaglandin synthesis through inhibition of cyclooxygenase-1 and cyclooxygenase-2 and suppresses leukotriene biosynthesis by inhibiting 5-lipoxygenase. These dual inhibitors of cyclooxygenase and 5-Lox consequently distinguished ginger to exert fewer side effects than non-steroidal anti-inflammatory drugs (GRZANNA et al., 2005;MASHHADI et al., 2013).
The high request on ginger worldwide as spice and reliable medicinal herb, especially with anti-inflammatory properties, is associated with increasing loss of ginger productivity when it is propagated under natural conditions in the field, because it is easily infected by many pathogenic factors including fungi, bacterial wilt, and nematodes. To overcome these problems, plant tissue culture was employed as an efficient technique to initiate microbial free plants or to induce masses of undifferentiated cells (callus tissue) in vitro as sustainable and steady sources of phytochemicals for industrial and commercial purposes. To increase the in vitro yield of bioactive components, elicitation represents the most effective strategy applied to rise the yield of bioactive secondary metabolites in different in vitro cultures.
It is well established that flavonoids and phenolic acids inhibit the activity of various cyclooxygenases and lipoxygenases (LAUGHTON et al., 1991). Conversely, PUFAs have the ability to control inflammation and leukotriene synthesis via affecting cyclooxygenases and lipoxygenases (ARAUJO et al., 2019). Moreover, PUFAs are known to inhibit arachidonic acid metabolism. A recent study showed that callus derived from ginger rhizome as well as callus treated by elicitors significantly suppress the LPS-induced production of TNF-α, IL-1 and IL-6 and production of the IL-10 and TGF-β anti-inflammatory cytokines (ALI et al., 2019). The present study, in continuation to evaluate the anti-inflammatory capacity of ginger callus, was undertaken to assess the 5-Lox inhibitory effect of ginger rhizome, callus and callus elicited by yeast extract, glycine and salicylic acid and to investigate their chemical profile by GC/MS analysis.

Plant materials
Ginger rhizomes were obtained from the botanical garden at Biology and Biotechnology Department, Faculty of Science and Technology, Al-Neelain University, Khartoum, Sudan. Rhizomes were well washed, cut into thin slices, and dried at room temperature.

Callus initiation and proliferation
Ginger callus initiation and proliferation were previously described by ALI et al. (2016). The best callus fresh weight was developed and proliferated on MS medium augmented by 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D).

Preparation of extracts
The extracts of ginger rhizome and callus were prepared by maceration in petroleum ether and chloroform: methanol (CM) (1:1, v/v) for 72 h at room temperature (ALI et al., 2018).

Gas chromatography/mass spectrometry (GC/MS) analysis
Analysis of the chemical composition of ginger rhizome and callus extracts were performed by gas chromatography coupled to mass spectrometry (Model GC-MS-QP2010 Plus, Shimadzu, Japan). Separation was performed using Rtx-5MS capillary column (5% diphenyl-95% dimethylsilicone, 30 m × 0.25 mm × 0.25 µm) and a temperature program of 50 °C (1 min) ramped to 300°C (3 min) at 5 °C/min. Identification of compounds was based on comparison of mass spectra with the GC/MS system data bank (NIST 08 library), comparison with published data, and retention indices. The relative amount of each compound was expressed as percent peak area relative to the total peak area of the GC chromatogram.

Lipoxygenase assay
5-lipoxygenase assay was performed following the procedure described by FRUM & VILJOEN (2019). Briefly, 12.5 μL of extract was mixed with 50 μL of linoleic acid (0.003 g/10 mL) and made up to 1 mL with 0.1 M phosphate buffer with Tween (0.005%). To initiate the reaction, 1.5 μL of 5-lipoxygenase from soybean (0.054 g/mL) was added to mixture. The increase in absorbance at 234 nm was recorded for 5 min in a Shimadzu 160-UV spectrophotometer. Nordihydroguaiaretic acid was used as positive control. The % enzyme inhibition was calculated by the following equation: % = [(A0 -A1) / A0] x 100 where A0 was the absorbance of the control without extract and A1 was the absorbance of the sample.

Statistical analysis
Data were statistically analyzed using SPSS version 19. The 5-lox experiment were performed in triplicate and the results were expressed as mean ± standard deviation (SD) values. Significant differences between samples were analyzed using analysis of variance (ANOVA) and Duncan's multiple-range test (P < 0.05).

Extraction yield
Different extraction yields (percentage of mass of extract/mass of dry matter) of ginger rhizome and untreated and treated callus are depicted in table 1. Oils of rhizome and untreated callus yielded low amount than their respective CM extracts and with higher quantity of rhizome oil than that of the callus. Treatment of callus with different elicitors highly increased the yield of CM extracts but did not provide measurable oil content. The ranking order of CM extracts of the untreated and treated callus was in the following decreasing order: callus treated by SA50 mg/L (27.2%) > callus treated by YE500 mg/L (23.49%) > callus treated by YE100 mg/L (23.25%) > callus treated by GL100 mg/L (19.86%) > callus treated by GL200 mg/L (18.58%) > callus treated by GL300 mg/L (17%) > callus treated by YE300 mg/L (12.18%) > callus treated by SA100 mg/L (10.75%) > untreated callus (5.74%).

Anti-5-lipoxygenase activity
The anti-5-Lox activity of the oil and CM extracts of ginger rhizome and untreated callus was evaluated, and results are presented in figure 1-a. The highest anti-5-Lox activity was exerted by callus oil (58.33 ± 4.66 μg/mL) and its CM extract (87.68±7.32 μg/mL) respectively. In fact, the callus oil was significantly (P ˂ 0.05) more active than the positive control; Nordihydroguaiaretic acid (IC 50 61.25±1.02 μg/mL) and was 2.9-fold more active than the rhizome oil. Moreover, the CM callus extract exhibited significant (P ˂ 0.05) 1.7-fold higher anti-5-Lox activity than that obtained from of the rhizome CM extract and was comparable to that exerted by 6-shogaol (87.65 ± 4.35 μg/mL). Further, the callus was treated with elicitors (YE, GL and SA) in an attempt to increase its anti-5-Lox activity. Results of anti-5-Lox activity of CM extracts from treated calli are presented in figure  1-b. Generally, elicitors enhanced significantly (P < 0.05) the callus inhibitory effect on 5-Lox activity. Treatment of callus with YE recorded highest inhibition levels against 5-Lox activity compared to other treatments of elicitors. One hundred and 300 mg/L YE decreased the 5-Lox activity by 33.16% and 25.46% respectively. SA 50 mg/L inhibited the 5-Lox activity by 16% while treatment of callus with GL reduced it by 10.46% and 4.82% at concentrations 200 and 300 mg/L respectively.

DISCUSSION
Plant tissue culture is considered as an effective technique to produce microbial free plants and a steady source of bioactive molecules for industrial and commercial purposes. Moreover, elicitation was proven as an excellent strategy to improve the in vitro yield of bioactive components in cultures (ALI et al., 2018). In the present study callus was obtained from ginger rhizome to evaluate their 5-Lox inhibitory property and then callus   was treated with elicitors to determine their effect on callus yield, anti-5-Lox activity, and chemical profile. Treatment of callus with the three elicitors; YE, GL and SA highly increased the yield of callus CM extracts but not its oil content. Many previous studies reported the effects of elicitors on enhancing the yield and production of high-added value plant compounds (ALI et al., 2018;CAI et al., 2014;RAMIREZ-ESTRADA et al., 2016). Results of the anti-5-Lox activity of ginger rhizome and callus revealed that the callus oil displayed remarkable anti-5-Lox activity with IC 50 value (58.33±4.66 μg/mL) significantly (P ˂ 0.05) lower than that of the standard control Nordihydroguaiaretic acid (61.25±1.02 μg/mL). Even the CM extract of the callus showed remarkable anti-5-Lox activity with IC 50 value comparable to that exerted by 6-shogaol (87.65±4.35 μg/mL). Previous studies reported that, the anti-5-Lox activity of ginger rhizome is mainly attributed to its major pungent compounds 6-gingerol and 6-shogaol with the latter exhibited potent anti-inflammatory. They exert their anti-inflammatory activity through the suppression effect on leukotriene biosynthesis by inhibiting 5-Lox (EZZAT et al., 2018;FLYNN et al., 1986;GRZANNA et al., 2005;KIUCHI et al., 1992). Chemical profile of the oil and CM extract of the callus indicated the absence of 6-gingerol, 6-shogaol, zingiberene and gingerol which were known as the major compounds of rhizome (KAMALIROOSTA et al., 2013;KIZHAKKAYIL & SASIKUMAR, 2012;NAMPOOTHIRI et al., 2012). Instead, the callus oil was rich in fatty acids (87.43 %). The unsaturated fatty acids, oleic acid (31.11%) and linoleic acid (18.63%) representing 49.9 % of the total fatty acid content and the saturated fatty acid, palmitic acid represented 28.56%. Many studies have demonstrated that callus extracts produced large amounts of fatty acids (BERNABÉ-ANTONIO et al., 2015;JACOMINI et al., 2015). The differences in fatty acids compositions in the oil of callus tissues and their mother plant may be related to variations in the gene expression of different cells, which showed alterations in their metabolism (DA LUZ COSTA et al., 2015). The anti-inflammatory activity of fatty acids was reported by HENRY et al. (2002) where they found that oleic acids possessed COX-I inhibitory activities while linoleic and linolenic acids showed appreciable COX-I and COX-II inhibitory activities. Linoleic acid inhibited the COX and LOX pathways of arachidonate metabolism (SINGH & MAJUMDAR, 1997). However, palmitic acid was reported to have marginal COX-I and COX-II inhibitory activities (HENRY et al., 2002). Thus, it could be suggested that the high fatty acid contents of the callus might also play considerable role in its anti-5-Lox activity.
Treatment of callus with different elicitors increased the yield of callus CM extracts and significantly (P ˂ 0.05) improved its capacity to inhibit the 5-Lox activity by 4.82% to 33.16% according to elicitor used and its concentration. The highest effect was obtained from the treatment of the callus by YE at 100 and 300 mg/L respectively followed by 50 mg/L of SA. Callus treated by GL showed the least effect. The chemical profiling of CM extracts of callus elicited with elicitors was generally different and revealed an enhancement in the production of some bioactive compounds that were not detected in untreated callus. Plant cells in vitro, displayed physiological and morphological responses   to the elicitors that could induce or enhance synthesis of secondary metabolites in plant cells or tissue to ensure their survival, persistence, and competitiveness (NAMDEO et al., 2002). Furthermore, some of the identified compounds in the treated callus extracts were proven to possess anti-inflammatory properties like lupeol which was identified in the CM extracts of callus treated with YE300 mg/L (11.58 %) and SA50 mg/L (9.78 %). A study carried out by THIRUMALAISAMY et al. (2020) showed that lupeol exhibited anti-inflammatory activity against the five targets of inflammation: COX-2, MPO, TNFα, IL1β and IL6, respectively. Also, β-amyrin (6.81% from callus treated with SA50 mg/L) significantly inhibited PGE2, IL-6 secretion, and NF-κB activation (KRISHNAN et al., 2014). Moreover, a previous study showed a significant increase in the total polyphenolic content of CM extracts of ginger callus especially those treated with YE100 mg/L and SA50 mg/L suggesting that phenolic compounds could also contributed to the observed activity (ALI et al., 2018).

CONCLUSION
In conclusion, this is the first study on the anti-5-Lox activity and chemical profile of ginger callus and callus treated with YE, SA and GL as elicitors. Results showed that the oil extracted from the callus was rich in fatty acids and exerted anti-5-Lox activity higher than the oil extracted from the intact rhizome and standard control as well. Elicitors modified the chemical profile of the callus and ameliorated the anti-5-Lox activity of CM extract of the callus. Further study is warranted to determine the phytochemical(s) responsible for this current observed bioactivity; and consequently, could lead to the development of potential natural-based anti5-Lox agent from ginger callus.