Metabolite profiling of the medicinal herb Phyllanthus niruri L. under drought stress

Plant-specialized metabolites are unique sources for pharmaceuticals and industrially valuable biochemicals. Accumulation of these metabolites is common in plants stimulated by biotic or abiotic stressors and/or elicitors. One of the abiotic factors that influence the plant’s bioactive molecule is drought stress, which can be employed to stimulate the production of bioactive compounds in plants. Phyllanthus niruri is commonly used as a medicinal plant in many parts of the world for the treatment of various diseases because it contains bioactive compounds that have the potential to be antioxidants, antimicrobials, and anti-hepatoprotective. Our previous studies have demonstrated that drought stress up to 70% field capacity (FC) could increase crop production as well as phenol and flavonoid contents. This study aimed to analyze the metabolite profile of P. niruri, which was subjected to drought stress for various FC (Control, 85%, 80%, 75%, 70%, 55%). Using chromatography-mass spectrophotometry (GC-MS) analysis, forty-seven compounds were identified. Three compounds were detected in control and all treatments, whereas four compounds were only detected in drought-stressed plants. β-sitosterol, 9-tricosane, heneicosane, and hexacosane are the main compounds only detected at 80% FC. The clustering analysis revealed that drought stress induced distinct compounds compared to control. Moreover, 85% -75% FC presumably induces similar compounds as they were in the same cluster, while plants responded differently upon 70% FC and 55% FC. In conclusion, P. niruri synthesizes different bioactive compounds under different drought-stress conditions.


Introduction
Plants are a natural product that has been used for centuries to treat various diseases.Recently, pharmacognostic, phytochemical, and pharmacological studies of traditional medicinal plants have received considerable attention.In addition, the biological activity of medicinal plants has been 2 investigated in many preclinical and clinical studies, revealing the diverse biological effects of a wide variety of plant-derived compounds from various chemical classes.However, the complexity of chemical composition in bioactive compounds from medicinal plants are currently an obstacle to the development of herbal medicines due to unclear bioactive compounds, mechanisms of action, etc. [1].Consequently, metabolomics, can be one of the advanced tools for enhancing and approaching new drug discovery to maintain the sustainability of herbal medicine [2].The metabolite profile of medicinal plants for new drug discovery can be analysed using the metabolomics technique of gas chromatography-mass spectrometry (GC-MS).Plants, as sessile organisms, have developed complex metabolic systems that generate a plethora of specialized metabolites to survive in harsh environments.Decades of research have revealed the genetic and biochemical basis for an array of specialized metabolic pathways in plants [3].Specialized metabolites play a significant role in the ecology and fitness of a species, often being restricted to specific taxa, hence the term specialized metabolites [4].Typically, plants exposed to multiple elicitors or signal molecules synthesize several compounds.Specialized metabolites are essential for the adaptation and survival of plants.Different environmental conditions such as high or low temperatures, drought, alkalinity, salinity, ultraviolet radiation damage, and pathogen infection could affect the plants and elicit the production of plant specialized [5].Drought stress inhibits the growth of medicinal and aromatic plants, yet plants adapt to biotic and abiotic stresses by producing specialized metabolites.Through receptors and sensors, plants respond to threat signals to stabilize against these stresses [6].Elicitation, or the stimulation of the production of specialized metabolites, ensures the plant's continued viability, resistance to environmental stresses, and survival.In contrast, specialized metabolites derive from plants such as volatile oils, flavonoids, alkaloids, glycosides, tannins, resins, etc used by humans for industrially important food additives, flavors, and medications.Accordingly, elicitation research can be conducted to increase the production of specialized metabolites for the pharmaceutical and drug industries.Phyllanthus niruri is a widely used herb of the Euphorbiaceae family in various traditional and medicinal systems.This plant is commonly used in various parts of the world, including Asia and America, as well as Indonesia, to treat a variety of diseases, such as a diuretic, antiviral, antiinflammatory, and hepatoprotective agent [7].Research on the genus Phyllanthus reveals that water stress increases the production of several beneficial specialized metabolites [8] and exposure to short-term drought stress increased specific specialized metabolites, such as corilagin, as an acclimatization response to drought stress conditions [9].Our previous drought stress study on P. niruri showed that 80% field capacity (FC) produced higher fresh weight, flavonoids, phenols, and yield than controls.There is limited data on metabolite profiling of P. niruri as a response to drought stress to explore compounds with the pharmacological activity that are useful for human health.Therefore, this study aimed to assess the effects of drought stress on the metabolite profiling of P. niruri.

Drought stress treatment
The seeds of P. niruri originate from Wuluhan, Jember.Three grams of P. niruri seeds were planted in 40 cm x 40 cm polyethylene bags containing a mixture of red soil, rice husk, and farmyard manure (1:1:1).Plants were arranged in a randomized block design with three replications and six different drought stress treatments as field capacity (Control, 85%, 80%, 75%, 70%, and 55%).According to Sarker & Oba, drought stress was implemented with modifications [10].Plants were watered at 100% field capacity until harvest, which occurred 7 weeks after planting.Furthermore, irrigation stops are based on the respective field capacity treatment.The field's capacity was determined by the gravimetric method and a soil moisture meter.

Sample extraction
The extraction of P. niruri follows the modified procedure described in [11].The plant samples were washed, dried at 40 o C for 7 days, and crushed.400 mg of the powder was macerated with 15 mL of methanol, and then sonicated at 40 o C for 15 minutes.The extract was filtered using Whatman filter paper no 1, and the filtrate was collected.Methanol was evaporated using evaporation at a temperature of 20 o C, 100 rpm, and then the remaining residue was dissolved using n-hexane.

GC-MS analysis
GC-MS analysis of P. niruri extracts was performed using GC Agilent 7890B (GC), and Gas chromatograph interfaced to a mass spectrometer, 5977A (MSD) equipped with an Agilent column type 19091S-433: 93.92873 DB-5MS UI 5% Phenyl Methyl Silox (30 m x 250 μm x 0.25 μm).Helium gas was used as the carrier gas at a constant flow rate of 1 mL/min and an injection volume of 1 μL.The temperature was programmed from 40 °C with an increase of 10 °C/min to 300 °C.Each component's relative amount (%) was calculated by comparing its average peak area to the total area.The obtained chromatograms were combined and sorted into the appropriate alignments.Estimates of retention times and precision mass spectral data were used to match the chemical components with the existing Wiley MS libraries 2008.Using RStudio, the GC-MS data is visualized as a heatmap.The dendrogram was created using a UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering algorithm.

Result
Each sample from the various drought stress treatments was shown on a heatmap of specialized metabolites profiles (Figure 1).GC-MS detected 47 specialized metabolites, yet only three compounds, 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione, nonacosane, and tetracosamethyl-cyclododecasiloxane were detected in all treatments, including in control.Four compounds were only detected at all levels of drought stress treatment such as triacontanal, dl-αtocopherol, 1-tricosane, dan 2,5-dihydroxybenzoic acid, indicating that other compounds were detected in specific drought treatments.Several compounds were only detected under certain drought stress treatments, such as β-sitosterol, 9-tricosene, heneicosane, and hexacosane, which were only detected at 80% field capacity.We constructed a dendrogram to compare the specialized metabolites profiles of different drought stress treatments.According to the dendrogram, the profile of secondary compounds after exposure to drought stress is different compared to the control (Figure 2).Drought stress on 85% FC until 75% FC tend to be grouped in the same cluster, while 70% FC and 55% FC are in other clusters.We hypothesized that P. niruri gave the same response by synthesizing the same compound on 85% FC until 75% FC drought treatment but gave a different response in the 70% FC and 55% FC drought stress treatment.Based on GC-MS results, the isolated specialized metabolites are mostly from alkadiene, alkanes, chromanol, esters, fatty acid, fatty alcohol, fatty aldehydes, phenol, glycol ether, ketone, organosilicon compounds, steroid, terpenoid, and triterpenoid (Table 1).A total of 47 compounds were identified from the GC-MS analysis of the methanol extract of P. niruri exhibiting various phytochemical activities.Phytochemical screening of the methanol extract of P. niruri revealed the presence of phyto-compounds that have been documented to have antioxidant and other activities.Alkadiene has antioxidant activity.Alkanes have antioxidant, anticancer, analgesic, antiinflammatory, antimicrobial, antibacterial, and antituberculosis activities.Chromanols have antiviral and antioxidant activities.Esters have a neurological effect, anti-atherosclerotic, antifibrotic, anti-ageing, anti-ulcer, and antifouling activities.

Discussion
Plants produce certain compounds to protect themselves from the effects of drought stress.Four compounds were detected at all levels of drought stress treatment such as triacontanal, dl-αtocopherol, 1-tricosane, dan 2,5-dihydroxybenzoic acid.Triacontanal reduces the effects of drought stress by increasing antioxidant enzymes, photosynthetic rates, and water use efficiency in leaves [12].dl-α-tocopherol increases antioxidant enzymes and proline accumulation [13].Another study stated that 1-tricosane increased in drought stress treatment [14], and 2,5-dihydroxybenzoic acid was detected in drought stress treatment [15].Several compounds were only detected in certain drought stress treatments, such as β-sitosterol, 9tricosene, heneicosane, and hexacosane, were only detected at 80% field capacity.Similarly, previous research also showed that the accumulation of tricosane, hexacosane and sterols increased due to drought stress treatment [14].β-sitosterol was the main phytosterol that increased proportionally on increasing levels of drought stress.Phytosterols could scavenge the oxidation of ROS produced by stress in the cell membrane.Phytosterols are also involved in controlling metabolic processes such as regulation of membrane permeability, fluidity, and signal transduction for cell division.They are involved in the activity of membrane-bound enzymes [16].Hexacosane was identified in leaf cuticular wax.The chemical characteristics of the cuticular wax and the increase in wax are the main determinants of plant cuticle permeability.Cuticle synthesis is influenced by genetics and the environment under certain conditions [17].According to low-water conditions, plants synthesize various lipid substances for detoxification and protection of pathogens, such as heneicosane and substances for drought tolerance mechanisms [18].According to produced compounds, dendrogram analysis indicated that different water levels significantly affect the specialized metabolites production in P. niruri.Interestingly, based on the similarity of the synthesis compounds, the drought stress treatment was grouped into three clusters.We hypothesized that P. niruri responded similarly to drought stress of 85% FC-75% FC by synthesizing the same specialized metabolites compounds, in contrast to the compounds synthesized as a response to drought stress of 70% FC and 55% FC.Specific drought stress conditions can induce the synthesis of certain specialized metabolites in P. niruri [9].The most compounds identified for drought stress treatment were alkenes.The high content of alkanes found in the methanolic extract of P. niruri indicated that alkanes are the common constituent as the response to drought stress treatment.The alkanes were the most dominant constituents in the wax composition of watermelon under drought-stress conditions [19].Fatty acid, chromanol, phenol, organosilicon compounds are commonly identified as a response of P. niruri to drought stress treatment.These compounds are the constituents of polymer cutin and cuticular waxes, commonly known as cuticle, which plants need to survive drought stress [20].Specialized metabolites compounds synthesized in drought stress treatment have pharmacological activities.Tetracosane has pharmacological potential as an anticancer [21].β-sitosterol, 9-tricosane, heneicosane, and hexacosane were only identified on 80% FC with potency as blood cholesterollowering agents [22] and antimicrobial [23].9-Hexacosene and dodecanoic acid, dodecyl ester, which only identified on 75% FC, have analgesic, anti-inflammatory [24], and antiseizure [25] activities.2,5-dihydroxybenzoic acid has antibacterial, antiviral, neurological effect, antiatherosclerotic, antifibrotic, anti-ageing, anti-ulcer, and anticancer activities [26], and lanosta-8,24dien-3-one has anti-fungal activities [27] identified that only on 70% FC.Dotriacontanal, nonadecyl trifluoroacetate, and octacosanal have only detected on 55% FC.Dotriacontanal act as anticonvulsant effects of epileptic seizures [28], nonadecyl trifluoroacetate act as antimicrobial [29], octacosanal act as antinociceptive, anti-inflammatory [30].These results indicate that drought stress treatment on P. niruri could be used to enhance beneficial bioactive compounds for human health.

Conclusion
According to the study above, metabolic productions of P. niruri, which is characterized by GC-MS analysis and chemical diversity analysis, are influenced by drought stress conditions.β-sitosterol, 9-tricosane, heneicosane, and hexacosane are only detected at 80% FC.The clustering analysis revealed that drought stress induced distinct compounds compared to the control.Moreover, 85%-75% FC presumably induces similar compounds as they were in the same cluster, while plants responded differently upon 70% FC and 55% FC.

Figure 1 .
Figure 1.Heatmap of the identified compound on various drought stress treatments of P. niruri.

Figure 2 .
Figure 2. The dendrogram of P. niruri was constructed based on specialized metabolites detected from various drought stress treatments.Different colour line showed the group of compounds from different field capacity (FC) treatment, blue line: control, tosca line: 85% FC, 80% FC, 75% FC, yellow line: 70% FC, orange line: 55% FC.
Fatty acids have antiseizure and antitumor activities.Fatty alcohol has antinociceptive activity.Fatty aldehydes have anticonvulsant effects on epileptic seizures and antinociceptive activities.Phenol has anti-enterococcal, antihyperglycemic, antihypertensive, and antiplatelet activities.Ketones have anti-fungal and antibacterial activities.Organosilicon compounds have anti-fungal, antioxidant, and antimicrobial activities.Steroids have blood cholesterol-lowering agents and antihyperglycemic activities.Triterpenoids have anticancer, antioxidant, and antimicrobial activities.Several compounds were only identified in certain drought stress treatments.Tetracosane was identified only in the drought stress treatment of 85% FC.

Figure 3 .
Figure 3. Phenotypes of P. niruri under different drought stress treatments.Seven-week-old plants were subjected to drought stress by withholding watering according to the field capacity (FC) treatment.Control plants were daily watered all the time.Photographs were taken at the end of the stress treatment.

Table 1 .
Detection of the secondary compound from P. niruri on different drought stress treatments by GC-MS.