Headspace SPME-GC-MS Analysis and in silico Molecular Docking Studies of Phytochemical Compounds Present in Houttuynia cordata Thunb

Aim: The study aimed to identify the phytochemical constituents present in the leaves, stems, and roots of H. cordata and perform in silico molecular docking studies of selected common compounds present in all three parts. Materials and Methods: The phytochemical components were investigated using the headspace solid-phase micro-extraction followed by gas chromatography-mass spectrometry and molecular docking was performed using Autodock Vina v.1.2.0. Results: β-pinene was found to be the major compound present in stem (73.89%), leaves (66.46%) and roots (42.88%). Four category targets were used for in silico molecular docking of 14 common compounds found in leaves, roots, and stems. The present study showed that the compounds caryophyllene and dihydro-cis-alpha-copaene-8-ol had antibacterial, antioxidant, anti-cancer, and anti-inflammatory activities. Conclusion: This work demonstrated the great utility of headspace SPME-GC-MS for the investigation of aromatic chemicals in a variety of edible and medicinal spices. With in silico molecular docking, we may look into the potential pharmacological activity of various volatile organic compounds present in H. cordata .


INTRODUCTION
Indigenous foods not only have a distinct flavor and taste of their own, but they also offer dietary variety and ensure the security of the household's food supply.The diets of the northeastern peoples of India are primarily composed of natural and traditional foods.H. cordata, locally referred to as "Toningkhok," is one such native food plant of Manipur.H. cordata is a perennial, aromatic herb belonging to the family Saururaceae, a fragrant medicinal plant with spreading rootstock.The Manipuri people use the herb both raw and cooked, and the local healer (Maiba) uses it to treat anaemia, gastritis, and tuberculosis as well as dysentery, muscular sprains, and stomach ulcers.Alkaloids, essential oils, flavonoids, and other chemical components with distinctive therapeutic effects are among the chemical ingredients found in H. cordata.In Manipur's plain and hill districts, the plant grows wild and is harvested for market sale.In Meghalaya, it is used in salads or cooked with other vegetables. 1 Leaf juices are used to cure cholera, dysentery, treat blood deficiencies, and blood purification. 2Naga tribe of Kiphire District, Nagaland uses the whole plant to cure stomach ache, cholera, and dysentery and as diuretic.It is also applied to skin diseases. 3Apatani Tribe in Arunachal Pradesh used the shoot of H. cordata for freshness, good sleep, and heart disorders. 4In the Senapati district of Manipur, roots and leaves are used to cure measles, gonorrhea, and skin troubles. 5In Southeast and East Asian countries, it is frequently used as an herbal anti-inflammatory, antibacterial, antiviral, and anti-cancer medicine.H. cordata has the potential for both antiviral activity and antiviral consequences, which is relevant given the recent COVID-19 pandemic reported by Lau et al. (2008) during the SARS outbreak of 2002-2003. 6][9][10][11][12] Due to its quick and ease of use, Headspace SPME is useful in the identification of plant volatile profiles.In addition, it acts as a tool to distinguish the phytochemicals present in different parts of the plant of the same species or different species.

Sample Collection and Preparation
The fresh portions of H. cordata were collected in September 2022 from Takyelpat, Imphal West District, Manipur.The plant was identified and authenticated by Botanical Survey of India, Sikkim Himalayan Regional Centre with voucher number IBSD-SC/ EPS/2020/IP/123.The authenticated herbarium was submitted in the IBSD Herbarium Library.The three parts of H. cordata viz., fresh leaves, roots, and stems were packed in a 15 mL clear vial which has a screw-top hole cap with silicone septa.All the vials were filled with one-third portions of the sample.The roots and stems were cut into identical lengths of 2 cm.

Headspace SPME-GC-MS
The SPME fiber was conditioned in the GC-MS and confirmed with no impurities through the blank run.The 15 mL glass vial containing the sample was exposed to the SPME fiber for 20 min.Each sample after the exposure was injected into the inlet injector of the GC-MS instrument for 2 min.Trace 1300 GC fitted with TG-5MS column and mass detector (TSQ DUO) with triple Quadropole was used to study the volatile organic compounds.Helium was used as the carrier gas, with a flow rate of 1 mL/ min, and a split ratio of 1:20 was maintained.The initial column temperature was programmed from 40ºC for 1 min to 250ºC at a rate of 5ºC per min, and then to 250ºC for 20 min. 13,14The relative VOC's constituents were expressed by its peak area percentage.Based on the comparison of mass spectra with those of the 2017 National Institute of Standards and Technology (NIST) GC-MS Libraries the volatile compounds were identified.

3D structure retrieval of protein targets
The Anti-Microbial (AM), Anti-Oxidant (AO), Anti-Cancer (AC), and Anti-Inflammatory (AI) targets were selected based on their prominence in the literature and their 3D structure was retrieved from Protein Data Bank (https://www.rcsb.org/).

Protein and Ligand preparation
Auto Dock MGL Tools v.1.5.7 was used for the protein preparation step. 17The identical chains, the native co-crystallized ligands, and the water molecules were deleted at first, the protein structure was added with polar hydrogen atoms and the Gasteiger charges were introduced.For the preparation of ligand molecules Chimera v.1.1.6software was utilized, the ligands were protonated and the charges were added. 18For the file format conversions Openbabel tool was used. 19

Binding site prediction
The binding sites of Antimicrobial and Antioxidant targets were comprehensively documented in previous research were used. 20or Anticancer and Anti-inflammatory targets, the binding site grid coordinates were predicted by using BIOVIA Discovery Studio, at first the co-crystallised ligands were selected and the "Define and Edit Binding Site" option was used. 21

Molecular docking and analysis
Autodock Vina v.1.2.0 which uses the Lamarckian algorithm was used for performing molecular docking. 22The native co-crystallized ligands and the short-listed phytochemical compounds were docked individually using the command line.For saving the docked ligand and protein complexes Pymol software was used. 23The images of active pocket visualization and 2D amino acid interaction between the ligand-protein complexes were obtained from BIOVIA Discovery Studio.

Molecular docking analysis
The Binding site grid coordinates used for molecular docking is listed in Table 4.The molecular docking of 14 phytochemical compounds with various targets was performed.The binding energies of all the compounds are represented as heat map as shown in Figure 2. The compounds Caryophyllene and dihydro-cis-alpha-copaene-8-ol were highlighted exclusively in this study as both of them had binding affinity to a higher number of targets compared to other compounds.Table 5 contains the binding energies and amino acid interactions of phytochemical compounds Caryophyllene and dihydro-cis-alpha-copaene-8-ol against various targets.
The compounds caryophyllene and dihydro-cis-alpha-copaene-8-ol showed high binding affinity of -7.7 and -7.8 kcal/mol against the antimicrobial target dihydrofolate reductase of S. aureus (PDB-ID: 3SRW).The enzyme Dihydrofolate Reductase (DHFR) is responsible for the NADPH-dependent conversion of dihydrofolate to tetrahydrofolate. 24Tetrahydrofolate is essential for several biosynthetic pathways, including amino acid and nucleic acid metabolism. 25DHFR inhibitors are effective for treating bacterial, mycobacterial, fungal, and protozoal infections hence we chose DHFR as an anti-microbial target. 25Caryophyllene interacted with the target DHFR with 9 van der Waals interactions and 9 Pi interactions.dihydro-cis-alpha-copaene-8-ol interacted with 1 hydrogen bond, 4 van der Waals interactions, and 10 pi interactions.The amino acid interactions of the compounds to the anti-microbial target 3SRW are shown in Figure 3.
Both compounds showed high binding affinities of -7.4 and -7.1 kcal/mol against the antioxidant target cytochrome P450 CYP2C9 (PDBID: 1OG5).Cytochrome P450 enzymes produce reactive oxygen species which provide oxidative stress, inhibiting this enzyme produces an antioxidant effect. 26,27Caryophyllene interacted with the target CYP2C9 with 4 van der Waals interactions and 10 Pi interactions.dihy-dro-cis-alpha-copaene-8-ol interacted with 1 hydrogen bond, 11 van der Waals interactions, and 10 pi interactions.The amino acid interactions of the compounds to the antioxidant target 1OG5 are shown in Figure 4.
The compounds showed higher binding affinities of -8.5 and -8 kcal/mol against the anticancer target Human Estrogen Receptor Alpha (PDBID: 3ERT) than other anticancer targets.The Estrogen Receptor alpha (ERα) is known to play an important role in cell proliferation in Breast cancer hence we used ERα as a potential anti-cancer target. 28,29Caryophyllene inter-acted with the target 3ERT with 7 van der Waals interactions and 8 Pi interactions.dihydro-cis-alpha-copaene-8-ol interacted with 6 van der Waals interactions and 9 pi interactions.The amino acid interactions of the compounds to the anticancer target 3ERT are shown in Figure 5 (A).
Though the compounds were having binding affinity to other anti-inflammatory targets, TNF-alpha was particularly chosen due to its extensive usage in the literature as an anti-inflammatory     target, making it an ideal selection for this study.The compounds showed high binding affinities of -7 and -7.8 kcal/mol against the anti-inflammatory Target Tumor Necrosis factor (TNF-alpha) (PDBID: 2AZ5).TNF-alpha is a pro-inflammatory cytokine that plays a major role in the pathogenesis of several inflammatory diseases.1][32] Caryophyllene interacted with the target TNF-alpha with 10 van der Waals interactions and 7 Pi interactions.dihydro-cis-alpha-copaene-8-ol interacted with the target 3SRW with 10 van der Waals interactions and 5 pi interactions.The amino acid interactions of the compounds to the anti-inflammatory target 2AZ5 are shown in Figure 5 (B).

DISCUSSION
β-pinene which was present in the leaves, roots, and stem of H. cordata is a well-known representative of the monoterpenes group, and is found in many plants' essential oils.There have been reports of a wide range of pharmacological activity, including the modulation of antibiotic resistance and analgesic, anticoagulant, anticancer, and antibacterial, effects that are anti-leishmanial, anti-inflammatory, anti-malarial, and antioxidants.β-pinene is used as an antibacterial due to its toxic effects on membranes and has been found to have inhibitory effects on leukemia and breast cancer.Leaf alcohol and leaf aldehyde are responsible for the green odor in leaves and fruits.1][32] α-Pinene is a monoterpene that is known to possess antimicrobial, apoptotic, anti-metastatic, and antibiotic properties.α-pinene is one promising agent for the treatment of various inflammatory diseases as it has been found to suppress MAPKs and the NF-κB pathway. 335][36][37][38] Camphene is a cyclic monoterpene that has anti-viral, insecticidal, antinociceptive, and antioxidant properties. 39,402][43] 2-Norpinene, 3,6,6-trimethyl, a chemical compound present in the leaves of H. cordata have antifungal properties.Due to its many benefits and uses, including its popularity in Northeast India traditional cuisine, this plant offers positive health benefits.

CONCLUSION
This study showed that headspace SPME-GC-MS is a very useful tool for the analysis of aromatic compounds of several edible and medicinally useful spices.In silico studies further supported the antimicrobial, antioxidant, anti-cancer, and anti-inflammatory properties of caryophyllene and dihydro-cis-alpha-copaene-8-ol found in H. cordata.Monoterpenes as the major components found in the leaves, roots, and stem of H. cordata, are in agreement with the in silico results that include antimicrobial, antioxidant, anti-inflammatories, and anti-cancer properties.Given all the beneficial active components, H. cordata acts as an excellent edible genetic resource and its consumption as a diet can benefit greatly in human health and promotes natural product research.Despite the potential and presence of numerous significant compounds, more research on the therapeutic applications of H. cordata can be performed.
Retention time; RSI b : Reversed search index on TG-5MS capillary column, RA c % : Relative area (peak area relative to the total peak area).

Figure 2 :
Figure 2: Heat-map representation of binding energies (in kcal/mol) of Phytochemical compounds against various targets.

Figure 3 :
Figure 3: Docking results depiction of the key amino acid interactions in the binding site of anti-microbial target DHFR (PDBID: 3SRW)with Caryophyllene and dihydro-cis-alpha-copaene-8-ol.

Table 1 : Major volatile organic compounds present in Houttuynia cordata leaves.
Headspace SPME-GC/MS Analysis and Molecular Docking of Phytochemicals in Houttuynia cordata Thunb a : Retention time; RSI b : Reversed search index on TG-5MS capillary column, RA c % : Relative area (peak area relative to the total peak area).Biona, et al.:

Table 4 : Binding site grid coordinates of targets used for molecular docking.
Biona, et al.: Headspace SPME-GC/MS Analysis and Molecular Docking of Phytochemicals in Houttuynia cordata Thunb