Effects of melatonin on plant secondary metabolite production.pptx

This PPT presents the effects of melatonin on secondary metabolites production , especially essential oils, in aromatic plants.

1. Evaluation of the Effects of indole amine derivative hormones on Essential oil production in Ocimum kilimandscharicum Guerke . Prepared by: Rabia Ayoubi Dep. Of Pharmacognosy , FoPh , KU.

2. Table of Contents Introduction Objectives Historical Background Methodology Result Discussion Conclusion and Recommendations References 2 8/25/2024

3. Introduction Plant growth and development is a complex process, which affected by numerous factors. Availability of nutrients and growth regulators, environmental conditions, biogeography, genetic variability, are some examples of the well-known variables which influence plant growth and development (Arnao & Hernandez-Ruiz, 2014) . Plants naturally have different strategies to fight damage induced by different stress factors. There are plenty of compounds and metabolites, which involved in plant stress responses. Ethylene (Dubois et al., 2018), nitric oxide (Domingos et al., 2015), hydrogen sulfide (Li et al., 2016), calcium (Gilroy et al., 2016), phytohormones, such as jasmonic acid (Martinez-Medina et al., 2016), gibberellic acid, abscisic acid (Li et al., 2016) and secondary metabolites (Mazid M, 2011) including essential oils are some examples of the stress-induced compounds produced by plants. 3 8/25/2024

4. Elicitors and Elicitation Elicitor is a substance which, when introduced in small concentrations to a living cell system, initiates or improves the biosynthesis of specific compounds. Elicitation is the induced or enhanced biosynthesis of metabolites due to addition of trace amounts of elicitors. Elicitors are classified on the basis of their nature (biotic & abiotic) or their origin (exogenous & endogenous). 8/25/2024 4

5. Melatonin as a biotic plant elicitors Melatonin (C 13 H 16 N 2 O 2 ); so called N-acetyl-5-methoxytryptamine and Circadin, is a potent indolamine neuro-hormone, which controls the circadian activities including sleep, blood pressure, and reproduction in a particular season. This compound is associated with plant stress responses, such as salinity, drought, cold, oxidative and nutritional stresses as well as plant development including root regeneration, growth, senescence, and flowering. 8/25/2024 5

6. Secondary metabolites S econdary metabolites (SMs) are those compounds, which have no known role in the basic life processes in the plants that produce them, and have role in the interaction of the plants with surroundings. Production of these molecules is often low and depends on the physiological and developmental stage of plants. Advancement of biochemical and molecular biological tools has been clearly shown that SMs play key role in the: Survival of plants in their environment Protection of plants from pathogens, competitors, herbivores etc. Protection of plants from irradiation Metabolic intermediates 6 8/25/2024

7. Essential oils Essential oils (EOs) are steam-volatile or organic-solvent extracts of plants traditionally used as deodorant, flavoring agents and preservative for many centuries. EOs are plant-derived chemicals and well known for their role against fungus, insect, and bacteria. EOs comprise mainly monoterpenes , the cyclic hydrocarbons and their alcohol, aldehyde or ester derivatives. These compounds are received more attraction due to their role as natural alternatives to chemical biocides and sometimes considered as antibiotics. 8/25/2024 7

8. Objectives To evaluate the effect of melatonin on: EO yield in Ocimum kilimandscharicum Guerke leaves Chemical profile of EO of Ocimum kilimandscharicum Guerke leaves Improving free-radical scavenging capacity of EO of Ocimum kilimandscharicum Guerke leaves 8 8/25/2024

9. Historical Background Melatonin, discovered for the first time from bovine pineal gland in 1995. Its primary biological function in antioxidant defense system, is studied in unicellular organisms (Reiter R. H.‐B., 2010). The presence of melatonin in Lingulodinium polyedrum (unicellular dinoflagellete) was detected thirty years after the discovery of melatonin in mammals. In 1993 melatonin was detected in Japanese morning glory and in 1995 in higher plants. Presence of melatonin in lichens is not known yet, and there is limited information about melatonin in bacteria, fungi, and non-vascular plants. Hardeland (2015) reviewed the occurrence and functions of melatonin in phototrophs from an evolutionary point of view and the place that melatonin deserves in the network of phytohormones . Reports obtained from Egypt showed the presence of serotonin, tryptamine, and melatonin in some edible as well as medicinal plants with different concentrations (Badria, 2004). 9 8/25/2024

10. Continue… Melatonin a well-known indoleamine, functions as neurotransmitter in mammals, shares the same precursor, tryptophan amino acid, with indole-3-acetic acid (IAA). This structural similarity with IAA makes melatonin as a candidate compound, which may function in the regulation of growth and development process in plants (Tan Q. S.‐X., 2016 ). The biochemical changes, which occur after melatonin treatment have been studied. Sarrou (2015 ) reported that different concentrations of melatonin could affect the essential oil, phenolics and flavonoids content in bitter orange ( Citrus aurantium L.) leaves. In addition to increase the total phenolic and flavonoid contents of leaves, melatonin treatment improved FRAP and DPPH activity of methanolic extracts of Citrus aurantium L leaves. The applicability of melatonin to increase the salt tolerance of plants has been explored. Application of melatonin (o.1 µM) in Malus hupehensis has improved the growth inhabitation and photosynthetic capacity (Li et al., 2018 ). In cucumber ( Cucumis sativus L.) plant grown under high salt concentration, the number of lateral roots was more than that of the control after melatonin treatment (Zhang et al., 2014). 8/25/2024 10

11. Ocimum kilimandscharicum Guerke 8/25/2024 11 Ocimum kilimandscharicum Baker ex Gürke is an evergreen perennial shrub with simple ovate to oblong leaves, light white-colored flowers, ovoid-oblong black or brown colored mucilaginous seeds. Plant is native to east African countries especially in Kenya, Tanzania, Uganda, Sudan, and Ethiopia. Due to its geographical distribution and abundance in African countries, it is commonly called as African blue basil. The EO, obtained from the leaves of this plant has light yellow color with distinct and strong scent of camphor. EO of this plant has antioxidant, anti-inflammatory and cancer controlling activity and the major constituents responsible for these effects are camphor, mixture of limonene, and 1, 8-cineol.

12. Methodology 8/25/2024 12 GCMS Analysis EO Chemical profiling Melatonin treatment In-Vivo Tests (foliar spray ) In-Vitro Tests (Plant Tissue Culture ) Hydro distillation (EO extraction ) EO quantification UV- Vis spectroscopy FTIR spectroscopy GCMS analysis Callus culture Nodal explant culture Whole plant treatment Solvent extraction Evaluation of plant growth Chemical profiling Folin - Ciocalteu Test LCMS analysi

13. EO yield percentage after melatonin treatment in two different time points Plant name Mentha x piperita L. Ocimum kilimandscharicum Guerke Time points (day) Concentration of applied melatonin ( mM ) EO yield % (v/w) Concentration of applied melatonin ( mM ) EO yield %(v/w) 0.7± 0.01 0.85± 0.07 2 0.1 0.58± 0.02 0.05 0.43± 0.02 0.4 0.22± 0.02 0.1 0.47± 0.02 5 0.1 0.39± 0.007 0.05 0.31± 0.01 0.4 0.15± 0.007 0.1 0.34± 0.02 8/25/2024 13

14. GCMS Analysis of EO GC chromatogram of EO of O. kilimandscharicum Guerke leaves obtained from control plant. GC chromatogram of EO of O. kilimandscharicum Guerke leaves obtained from O1 plant (0.05mM melatonin treatment and collected after 2 days ). 8/25/2024 14 Chemical profiling of EO of Ocimum kilimandscharicum Guerke leaves

15. GC chromatogram of EO of O. kilimandscharicum Guerke leaves obtained from O2 plant (0.05mM melatonin treatment and collected after 5 days ). GC chromatogram of essential oil of O. kilimandscharicum Guerke. leaves obtained from O3 plant (0.1mM melatonin treatment and collected after 2 days) . 8/25/2024 15

16. GC chromatogram of EO of O. kilimandscharicum Guerke leaves obtained from O4 plant (0.1mM melatonin treatment and collected after 5 days ). GC chromatogram of EO of O. kilimandscharicum Guerke. leaves obtained from O5 plant (0.5mM melatonin treatment and collected after 2 days) 8/25/2024 16

17. Percentage of the most abundant phytoconstituents in composition of EO obtained from leaves of O. kilimandscharicum Guerke after melatonin treatment. 8/25/2024 17 O1: 0.05mM melatonin treatment and collected after 2 days; O2: 0.05mM melatonin treatment and collected after 5 days O3: 0.1mM melatonin treatment and collected after 2 days; O4: 0.1mM melatonin treatment and collected after 5 days Compound name Percentage of the most abundant components in total oil C O1 O2 O3 O4 α- Pinene 0.55± 0.01 0.09± 0.01 0.23± 0.02 0.04± 0.02 0.21± 0.02 β- Ocimene 20.03± 0.02 8.43± 0.02 11.47± 0.02 9.09± 0.02 13.47± 0.02 Germacrene -D 3.16± 0.02 0.42± 0.02 0.42± 0.01 8.31± 0.02 3.55± 0.03 Eugenol 70.32± 0.02 76.59± 0.02 79.18± 0.03 74.04± 0.03 78.08± 0.03 α- ylangene 0.51± 0.01 0.5± 0.2 0.21± 0.02 0.63± 0.02 0.27± 0.03

18. Percentage of the disappeared phytoconstituents in composition of EO obtained from leaves of O. kilimandscharicum Guerke after melatonin treatment. S. No. Compound name Percentage of the disappeared phytoconstituents in total oil C O1 O2 O3 O4 1 Terpinolene 0.19 2 o-Cymene 0.05 3 cis-α-Bisabolene 0.11 0.33 0.18 0.32 0.15 4 Patchoulane 0.04 0.06 0.04 0.32 0.04 5 Z-cis-α-Bisabolene epoxide 0.02 0.05 6 Caryophyllene oxide 0.13 0.04 7 Andrographolide 0.03 8 Decanexent 0.03 8/25/2024 18 O1: 0.05mM melatonin treatment and collected after 2 days; O2: 0.05mM melatonin treatment and collected after 5 days O3: 0.1mM melatonin treatment and collected after 2 days; O4: 0.1mM melatonin treatment and collected after 5 days

19. Percentage of the newly synthesized phytoconstituents in composition of EO obtained from leaves of O. kilimandscharicum Guerke after melatonin treatment. 8/25/2024 19 S. No. Compound name Percentage of the newly appeared phytoconstituents in total oil C O1 O2 O3 O4 1 (-)- Carvone 0.85± 0.03 0.07± 0.02 2 α- Cubebene 0.01± 0.02 3 cis-Muurola-3, 5-diene 0.25± 0.02 0.3± 0.02 0.13± 0.02 4 Isocaryophyllene 4.29± 0.02 2.02± 0.02 3.99±0.08 1.93± 0.03 5 cis- β- Farnesene 0.05± 0.02 6 Germacrene-D-4-ol 0.14± 0.02 0.24± 0.02 0.1± 0.02 7 cis- α- Bisabolene 0.11 0.02 0.33± 0.02 0.18± 0.02 0.32± 0.02 0.15± 0.02 8 Patchoulane 0.04± 0.02 0.06± 0.02 0.04± 0.02 0.32± 0.03 0.04± 0.02 9 Z-cis- α- Bisabolene epoxide 0.02± 0.01 0.05± 0.03

20. 8/25/2024 20 Percentage of the newly synthesized phytoconstituents in composition of EO obtained from leaves of O. kilimandscharicum Guerke after melatonin treatment . O1: 0.05mM melatonin treatment and collected after 2 days; O2: 0.05mM melatonin treatment and collected after 5 days O3: 0.1mM melatonin treatment and collected after 2 days; O4: 0.1mM melatonin treatment and collected after 5 days

21. GC-MS analysis of essential oils of Mentha piperita L . leaves GC chromatogram of EO of M. piperita L. leaves obtained from control plant . GC chromatogram of EO of M. piperita L. leaves obtained from control plant (0.1mM & 2 days ). 8/25/2024 21 Qualitative and Quantitative analysis of EO of Mentha x piperita L . leaves

22. Percentage of the most abundant components in composition of EO obtained from leaves of M. piperita L . plant before and after melatonin treatment . 8/25/2024 22 C: control; M1: 0.1mM melatonin treatment & collected after 2 days S. No. Compound name Percentage of the most abundant components in total oil C M1 1 β- Ocimene 0.4± 0.01 0.2± 0.02 2 Isomenthone 14.38± 0.02 14± 0.3 3 ¥-Terpineol 1.17± 0.02 1.7± 0.2 4 d-Menthol 70.99± 0.02 76± 0.3 5 L- α- Terpineol 0.24± 0.01 0.2± 0.02 6 Pulegone 0.35± 0.02 0.2± 0.02 7 Piperitone 0.44± 0.04 0.3± 0.07 8 α- Cadinol 0.17± 0.02 0.1± 0.01

23. Percentage of the phytoconstituentsin composition of EO obtained from leaves of M. piperita L. plant, which disappeared after melatonin treatment 8/25/2024 23 C: control; M1: 0.1mM melatonin treatment & collected after 2 days S. No. Compound name % of disappeared components in total oil C M1 1 α- Pinene epoxide 0.34± 0.02 2 Eugenol 6.56± 0.02 3 α- ylangene 0.18± 0.01 4 (4-Allyl-2-methoxyphenoxy) trimethylsilane 0.03± 0.02 5 β- ylangene 1.87± 0.02 6 Patchoulane 0.18± 0.02

24. Percentage of the newly synthesized phytoconstituents in composition of EO obtained from leaves of M. piperita L. plant after melatonin treatment. 8/25/2024 24 C: control; M1: 0.1mM melatonin treatment & collected after 2 days S. No. Compound name % of newly synthesized components in total oil C M1 1 Sabinene 0.1± 0.01 2 β- Pinene 0.3± 0.03 3 p-Menth-8-en-3-ol 0.4± 0.01 4 trans-Carveol 0.1± 0.01 5 (-)-Carvone 3± 0.03 6 β- Farnesene 0.1± 0.01 7 Bicyclogermacrene 0.01± 0.02

25. UV-Vis Spectra of EO of Ocimum kilimandscharicum Guerke leaves 8/25/2024 25 C O1 O2 UV Spectra of essential oil of O. kilimandscharicum Guerke leaves before and after melatonin treatment. O1: 0.05mM melatonin treatment and collected after 2 days; O2: 0.05mM melatonin treatment and collected after 5 days

26. 8/25/2024 26 O3 O4 UV Spectra of essential oil of O. kilimandscharicum Guerke leaves before and after melatonin treatment. O3 : 0.1mM melatonin treatment and collected after 2 days; O4: 0.1 2 mM melatonin treatment and collected after 5 days

27. 8/25/2024 27 S. No. Compound name Control O1 O2 O3 O4   OD  OD  OD  OD  OD 1 Poly-unsaturated and aromatic compounds 391.20 0.019 - - - - - - - - 2 - - - - - - 372.00 0.048 - - 3 368.00 0.024 369.40 0.024 365.60 0.069 - - - - 4 - - 357.80 0.024 - - 358.00 0.047 - - 5 - - - - - - 352.40 0.048 353.00 0.039 6 341.40 0.029 342.00 0.025 343.00 0.075 341.20 0.048 345.40 0.039 7 - - 332.80 0.025 331.80 0.097 - - 334.60 0.044 8 - - 328.60 0.026 - - - - 327.20 0.047 9 - - - - - - 317.80 0.083 - - 10 309.40 0.203 - - - - 310.20 0.100 309.80 0.083 11 287.60 2.750 287.40 2.568 286.40 3.037 288.40 0.948 284.00 3.042 12 - - - - 283.00 3.006 282.20 1.213 - - 13 276.00 2.976 278.60 2.929 279.00 3.031 - - 273.20 3.466 Table 17. The different wavelengths and absorbance of essential oil phytoconstituents obtained from leaves of O. kilimandscharicum Guerke leaves before and after melatonin treatment. O1: 0.05mM melatonin treatment and collected after 2 days; O2: 0.05mM melatonin treatment and collected after 5 days O3: 0.1mM melatonin treatment and collected after 2 days; O4: 0.1mM melatonin treatment and collected after 5 days

28. 8/25/2024 28 C=O, H-CH=O 15 269.80 2.943 - - 267.80 3.339 - - 268.40 2.805 16 C-X, CH3-OH, CH3NH2, CH3I 266.40 2.323 - - - - - - - - 17 249.80 3.268 - - - - - - 246.00 3.621 18 - - - - 241.60 3.433 - - - - 19 239.60 3.417 235.20 3.352 - - - - 237.00 3.506 20 224.60 3.277 222.20 3.153 - - 222.80 3.197 224.60 3.396 21 217.60 3.584 218.60 3.510 - - - - 218.00 3.704 22 - - - - 214.00 3.613 - - 212.60 3.531 23 208.80 3.474 200.40 3.558 - - - - 203.00 3.562 24 199.20 3.504 195.20 3.315 - - 197.00 3.677 196.20 3.182 25 - - - - - - 191.80 3.024 - -

29. . UV-Vis spectra of EO of M. piperita L. leaves 8/25/2024 29 Control M1 The different wavelengths and absorbance of essential oil phytoconstituents obtained from leaves of M. piperita L. leaves before and after melatonin treatment. M1: 0.1mM melatonin treatment & collected after 2 days

30. 8/25/2024 30 S. No. Chromophore  max(nm) OD C M1 C M1 1 Colored compound 393.20 - 0.018 - 2   Poly-unsaturated and aromatic compounds     - 371 - 0.008 3 369.80 - 0.024 - 4 352.60 353 0.024 0.008 5 345.00 342.20 0.023 0.006 6 - 331.60 - 0.004 7 - 316.20 - 0.009 8 - 306.80 - 0.018 9 - 298.20 - 0.045 10 290.80 292.40 0.512 0.140 11 287.00 283.20 0.662 -0.149 12 276.00 275.00 1.201 -0.071 Table 31. The different wavelengths and absorbance of essential oil phytoconstituents obtained from leaves of M. piperita L. leaves before and after melatonin treatment. M1: 0.1mM melatonin treatment & collected after 2 days

31. 8/25/2024 31 S. No. Chromophore  max(nm) OD C M1 C M1 13 C=O, H-CH=O(n-  *,  -  * transition) - 269.00 - -0.175 14 C-X (n-  * transition) 237.00 237.60 3.297 0.211 15 226.20 228.40 3.369 1.357 16 220.40 - 3.438 - 17 213.00 209.40 3.454 2.816 18 207.60 204.20 3.559 3.012 Table 31. The different wavelengths and absorbance of essential oil phytoconstituents obtained from leaves of M. piperita L. leaves before and after melatonin treatment. M1: 0.1mM melatonin treatment & collected after 2 days

32. FTIR spectra of essential oil of Ocimum kilimandscharicum Guerke leaves 8/25/2024 32 Control Eugenol O1 Figure 22. FTIR Spectra of essential oil of O. kilimandscharicum Guerke leaves before and after melatonin treatment. O1: 0.05mM melatonin treatment and collected after 2 days

33. Figure 22. FTIR Spectra of essential oil of O. kilimandscharicum Guerke leaves before and after melatonin treatment. O2 : 0.05mM melatonin treatment and collected after 5 days O3: 0.1mM melatonin treatment and collected after 2 days; O4: 0.1mM melatonin treatment and collected after 5 days 8/25/2024 33 O4 O2 O3

34. 8/25/2024 34 ID Bond assignment Control O1 O2 O3 O4 Eugenol 1 Strong C-X stretch - - - 441.71   441.71 2 - 497.65 - -     553.59 - 555.52 - 555.52 553.59 3 4 596.02 - 596.02 - 596.02 596.02 5 644.25 - 648.1 - 646.17 646.17 6 746.48 - 746.48 - 746.48 744.55 7 - - - - 792.77 - 8 =C-H-out of-plane bending (aromatic substitution) 804.34 - 804.34 - 817.85 804.34 9 - - 852.56 - 850.64 852.56 10 908.5 - 910.43 - 910.43 910.43 11 C-H-out-of-plane deformation 993.37 - - - 993.37 997.23 12 Strong C-OH/C-O-C stretch 1031.95 - 1031.95 1031.95 1033.88 1030.02 13         1120.68 - 14 1151.54 - 1151.54 - 1147.68 1136.65 15 - - - - 1205.55 - 16 1228.7 - 1228.7 - 1232.55 - 17 1267.27 - 1265.35 1267.27 - - 18 Medium CH3 bend 1367.58 - 1367.58 1369.5 136758 - 19 1437.02 - 1437.02 1437.02 1431.23 1437.09 20 - - - - 1458.23 - The different wavenumbers and absorbance of essential oil phytoconstituents obtained from leaves of O. kilimandscharicum Guerke leaves before and after melatonin treatment. O1: 0.05mM melatonin treatment and collected after 2 days; O2: 0.05mM melatonin treatment and collected after 5 days O3: 0.1mM melatonin treatment and collected after 2 days; O4: 0.1mM melatonin treatment and collected after 5 days

35. 8/25/2024 35 21 Weak C=C aromatic/ strong C=O amide 1510.31 - 1510.31 1512.24 1510.31 1510.31 22 Weak non-conjugated C=C stretch(alkene) 1606.76 - 1606.76 - 1608.69 1606.76 23 - 1631.83 - 1635.69 1637.62 - 24 C  stretch 2104.41 - - - - - 25 CH3 stretch 2357.09 2360.95 2357.09 2357.09 2360.95 2357.09 25 Weak -C-H stretch (aldehyde) - - - - 2845.1 - 27 Weak –C-H stretch 2928.04 - 2933.83 - 2933.83 2968.55 28 - - - - 2970.48 - 29 Weak =C-H stretching (sp 2 absorption) - - - - 3076.56 - 30 Strong H-bounded OH stretch (alcoholic OH)   3315.74 - 3338.89 - - 31 3524.06 3518.28   - 3520.21 3516.35

36. FTIR spectra of essential oil of Mentha piperita L . leaves 8/25/2024 36 Control M1 M2 Figure 35. FTIR spectra of essential oils obtained from leaves of M. piperita L. from control and melatonin-treated plants. M1: 0.1mM melatonin treatment collected after two days. M2: 0.1mM melatonin treatment collected after five days.

37. 8/25/2024 37 Table 32. A detailed explanation of different related wavenumbers and the corresponding functional groups of FTIR spectra of essential oils obtained from leaves of M. piperita L. from control and melatonin-treated plants. M1: 0.1mM melatonin treatment collected after two days; M2: 0.1mM melatonin treatment collected after five days S. No. Bond assignment Control M1 M2 1 Strong C-X stretch - - 503.44 2 545.87 545.87 648.1 3 - - 763.84 4 862.21 918.15 - 840.99 5 Strong C-OH/C-O-C stretch - 914.29 1035.81 914.29 977.94 6 1035.81 - 1020.38 7 1099.46 1093.67 1080.17 8 - - 1219.05 1298.14 9 Medium CH 3 bend 1269.2 1368.5 - - 10 - 1371.43 1371.43 11 1452.45 1454.38 1705.13 1446.66

38. 8/25/2024 38 12 C=C alkene - - 1600.97 1672.34 13 C=O amide/ketone/aldehyde 1705.13 - - 14 C  C - 2114.05 - 15 Weak -C-H stretch - - 2864.39 16 2933.83 3379.4 2920.32 2922.25 17 Strong N-H stretch - 3362.04 - 18 Strong alcohol OH stretch - - 3481.63

39. Anti-oxidant Activity (DPPH assay) S4 : 0.1mM melatonin treatment & collected after 5 days. S6: 0.2mM melatonin treatment & collected after 5 days. 8/25/2024 39 S. No Sample Anti-oxidant activity (DPPH assay) IC50 Value(µg/mL) 1 Ascorbic acid 0.27±0.03 2 Control 3.27±0.02 3 S4 1.61±0.05 4 S6 3.155±0.02 Control Melatonin-treated

40. S. No Sample Anti-oxidant activity (DPPH assay) IC50 Value(µg/mL) 1 Ascorbic acid 0.27±0.03 2 Control 7.77±0.02 3 O2 7.25±0.07 8/25/2024 40 Anti-oxidant activity of O. kilimandscharicum oil from leaves of control and melatonin-treated plants. O2: 0.05mM melatonin treatment and collected after 5 days

41. In-vitro studies 8/25/2024 41 Cultured nodal segments of M. spicata on MS medium containing BAP, melatonin and combination of BAP+ melatonin. A: control (MS medium without any growth regulator); B: MS medium containing 0.5mg/L BAP C: MS medium containing 0.5mM melatonin; D: MS medium containing 1mM melatonin E: MS medium containing combination of 0.5mg/L BAP+ 0.5mM melatonin F: MS medium containing combination of 0.5mg/L BAP+ 1mM melatonin A B C D E F

42. Total phenolic content Assay S. No. Concentration of melatonin in media(mg/mL) OD Concentration of phenolics (mg/g) mg/g% of total phenolics 1 control 1.610 70.30 374.31±0.04 2 0.1 2.193 95.72 703.86±0.01 3 0.2 0.741 32.35 188.29±0.06 4 0.3 1.038 45.29 391.43±0.06 5 0.4 1.447 63.18 451.27±0.05 6 0.5 0.615 26.85 228.09±0.04 8/25/2024 42 Percentage of total phenolic content in methanolic extract of M. piperita leaves in presence of different concentrations of melatonin.

43. In-vitro culture of Mentha piperita under stress condition 8/25/2024 43 Mel (0.1mM) NaCl (100mM) NaCl (100mM)+ Mel (0.1 mM HgCl2 (100mM)

44. UPLC –MS analysis of crude extract of M. piperita leaves cultured on MS medium in presence of 0.1mM melatonin UPLC chromatogram of crude extract of M. piperita leaves cultured on MS medium in presence of 0.1mM melatonin. MS spectrum of crude extract of M. piperita leaves cultured on MS medium in presence of 0.1mM melatonin . 8/25/2024 44

45. UPLC- MS analysis of crude extract of M. piperita leaves cultured on MS medium under salinity stress condition (in presence of 100mM NaCl) UPLC chromatogram of crude extract of M. piperita leaves cultured on MS medium under salinity stress condition (in presence of 100mM NaCl ). MS spectrum of crude extract of M. piperita leaves cultured on MS medium under salinity stress condition (in presence of 100mM NaCl ). 8/25/2024 45

46. UPLC chromatogram of crude extract of M. piperita leaves cultured on MS medium containing a combination of 100mM NaCl+ 0.1mM melatonin UPLC chromatogram of crude extract of M. piperita leaves cultured on MS medium containing a combination of 100mM NaCl+ 0.1mM melatonin MS spectrum of crude extract of M. piperita leaves cultured on MS medium containing a combination of 100mM NaCl+ 0.1mM melatonin 8/25/2024 46

47. UPLC chromatogram of crude extract of M. piperita leaves cultured on MS medium under heavy metal stress condition (in presence of 100Mm HgCl 2 ) UPLC chromatogram of crude extract of M. piperita leaves cultured on MS medium under heavy metal stress condition (in presence of 100Mm HgCl 2 ). MS spectrum of crude extract of M. piperita leaves cultured on MS medium under heavy metal stress condition (in presence of 100mM HgCl 2 ). 8/25/2024 47

48. A comparative table of percentage of different phyto constituents of methanolic crude extract of peppermint plant cultured on MS medium in presence of melatonin (0.1mM), NaCl (100 mM), combination of melatonin (0.1 mM) + NaCl (100mM) and HgCl 2 (100 mM). A: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of melatonin (0.1mM ) B: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of NaCl (100 mM ) C: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of combination of melatonin (0.1 mM) + NaCl (100mM) D: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of HgCl 2 (100 mM). 8/25/2024 48 S. No. RT Area Percentage A B C D 1 0.98 0.09 0.18 0.13 0.1 2 1.09 0.18 0.15 0.2 0.26 3 1.17 0.05 0.06 0.03 0.05 4 1.29 0.08 5 1.4 0.05 0.06 0.04 0.05 6 1.61 0.8 1.46 1.73 0.86 7 2.08 6.6 7.64 6.91 12.84 8 2.43 0.67 9 3.01 3.6 10 3.14 2.49 3.22 0.53 11 3.19 2.3 12 3.46 0.48 13 3.58 0.46 0.25 0.92 14 3.85 0.15 0.27 0.28 0.23 15 4.07 0.17 16 4.32 2.56 1.41 3.37 1.56 17 4.47 0.21 0.27 0.19 0.25 18 4.59 0.57

49. 8/25/2024 49 19 4.67 0.18 0.21 0.15 20 4.88 0.32 0.27 0.26 0.26 21 5.07 1.31 1.36 1.52 1.16 22 5.18 0.18 0.26 0.43 23 5.55 0.18 0.12 24 5.68 0.4 0.41 0.33 25 5.93 2.11 26 6.08 1.17 2.1 1.05 3.66 27 6.48 2.6 3.42 1.35 1.19 28 6.64 23.32 23.92 24.82 23.25 29 6.72 1.97 1.31 30 6.89 40.48 43.71 42.58 35.04 31 7.08 5.32 3.73 4.05 3.08 32 7.18 0.87 2.14 33 7.23 2.39 34 7.59 2.45 1.45 2.76 1.25 35 7.7 1.11 0.59 0.76 1.12 36 7.77 0.4 37 7.85 1.22 38 8.42 0.81 0.88 0.59 1.32 39 8.82 1.71 40 8.91 0.36 1.22 41 9.28 0.7 1.11 1.19 0.99 42 9.72 0.19

50. P ercentage of different phyto constituents of methanolic crude extract of peppermint plant cultured on MS medium in presence of melatonin (0.1mM), NaCl (100 mM), combination of melatonin (0.1 mM) + NaCl (100mM) and HgCl 2 (100 mM). A: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of melatonin (0.1mM) B: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of NaCl (100 mM) C: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of combination of melatonin (0.1 mM) + NaCl (100mM) D: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of HgCl 2 (100 mM). 8/25/2024 50

51. Percentage of different phyto constituents of methanolic crude extract of peppermint plant cultured on MS medium in presence of melatonin (0.1mM), NaCl (100 mM), combination of melatonin (0.1 mM) + NaCl (100mM) and HgCl 2 (100 mM). A: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of melatonin (0.1mM) B: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of NaCl (100 mM) C: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of combination of melatonin (0.1 mM) + NaCl (100mM) D: chemical composition of methanolic crude extract of peppermint plant cultured on MS medium in presence of HgCl 2 (100 mM). 8/25/2024 51

52. Conclusion As per objectives of this study and considering the results of all in-vivo and in-vitro experiments as well as the possible mechanisms, through which melatonin can influence the production of secondary metabolites in different aromatic plants, it is concluded that melatonin , a well know biogenic amine, which originally comes from pineal gland of animals and functions as a potent free-radicle scavenging molecule, circadian rhythm regulator, a neurologic bio-amine and etc., could be considered as: An efficient elicitor to induce biosynthesis of different metabolites in plants in a concentration and time-dependent manner. Change the oil yield percentage of aromatic plants Improve phyto-chemical profile of their volatile compounds qualitatively E nhance free-radical scavenging activity of plants’ essential oil I ncrease total phenolic content of plant crude extracts So it may act as a promising molecule to increase aromatic crops yield with desired quality. 8/25/2024 52

53. Further studies The experiments should be conducted in aromatic plants under controlled conditions (green house ). Before melatonin treatment, the genomic, transcriptomic and metabolomics data regarding phytomelatonin should be available. If possible, cloning of phytomelatonin genes would be performed. 8/25/2024 53

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