1. Introduction

OALibJ

Open Access Library Journal

2333-9705

Scientific Research Publishing

10.4236/oalib.1109750

OALibJ-131812

Articles

Biomedical&Life Sciences Business&Economics Chemistry&Materials Science Computer Science&Communications Earth&Environmental Sciences Engineering Medicine&Healthcare Physics&Mathematics Social Sciences&Humanities

Identification of Sec-Butylamine and Methenamine and Other Bioactive Compounds from Edible <i>Azadirachta indica</i> Methanolic Extracts Using GC-MS

Francis

Chukwuebuka Ihenetu

¹^*Sylvester

Ifunanya Okorondu

¹Chinwe

Ifenyinwa Chikwendu

¹Rose

Nkechinyere Nwabueze

¹Ifeoma

Ijeoma Anekwe

Department of Microbiology, Federal University of Technology, Owerri, Nigeria

07032024

110311712, January 202315, March 2024 18, March 2024

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Background: Azadirachta indica (Neem) is a medicinal plant that is traditionally known for its antimicrobial properties. The present study was aimed to determine the bioactive components of the A. indica leaves using GC-MS. Results: Results revealed presence of 35 different active compounds in different percentage composition successions. Bioactive compounds listed include; Methenamine, Hexadecanoic acid, methyl ester; Phenol; Dodecanoic acid, methyl ester; Methyl tetradecanoate; sec-Butylamine; 2-Propanone, 1-methoxy-; 10-Octadecenoic acid, methyl ester; Heptadecanoic acid, 16-methyl-; Phenol, 2,6-dimethoxy-; Phenol, 2-methoxy-; n-Hexadecanoic acid. Their molecular weight and formula was observed while their biological activities were also illustrated. Conclusions: The findings of this study suggest that A. indica leaf collected from the study area contain sec-Butylamine and Methenamine that have not been identified from other studies which are important bioactive compounds that can be used in several biological activities.

<i>Azadirachta indica</i> Neem GC-MS Bioactive Sec-Butylamine Methenamine

1. Introduction

Gas chromatography-mass spectrometry (GC-MS) is a well-established technology for identifying bioactive chemicals in medicinal plants, such as volatile matter, long chain and branch chain volatiles (BCVs). Gas chromatography with flame ionization detector (GC-FID) and gas chromatography mass spectrometry (GC-MS) are recommended for quantitative analysis [1] .

Azadirachta indica (neem) belonging to Meliaceae family is a very important medicinal plant which is traditionally used to treat different diseases. The evergreen tree neem (family Meliaceae, genus Azadirachta) is used to treat malaria in Nigeria and other African nations. It’s one of India’s most well-known plants, and it is grown in tropical and subtropical climates all over the world [2] . From antiquity, every component of the tree has been employed as a traditional medicine for domestic remedies against a variety of human maladies [3] .

Several studies have shown that neem contains a number of beneficial triterpenoids from the limonoids class. The primary non-volatile chemicals include Azadirachtin (Azadirachtin A), Nimbidiol, 3-tigloylazadirachtol (Azadirachtin B), Salannol, Salannin, Nimbinin, Nimbin, and 1-tigloyl-3-acetyl-11-hydroxy-meliacarpin (Azadirachtin D) [4] . Neem also contains sulfur-modified fatty substances such loeic acid (50% - 60%), palmitic acid (13% - 15%), stearic acid (14% - 19%), linoleic acid (8% - 16%), and arachidic acid (1% - 3%).

A visit to various herbalists in Imo State, Nigeria revealed a recognized and fascinating consistency in their regular usage of indigenous medicinal herbs for the treatment of skin and wound infections such as Erythrasma, impetigo, ecthyma, folliculitis, erysipelas, and cellulitis (which are generally known to the herbalist as Ocha-ere). The bioactive constituent of this plant in the region were reached were investigated to determine the variety of compounds it posses and their medicinal importance using GC-MS methods.

2. Methods2.1. Sample Collection and Preparation

Fresh leaves of Azadirachta indica were collected from farms from the study area. The plants were identified, validated and recognized by a Plant Taxonomist at Imo State University, Department of Crop Science, based on their taxonomic classification and further deposited in the herbarium Department of Crop Science, Imo State University Owerri. The leaves were air dried for 6 days at 27˚C. The dried leaves were pulverized into powder.

2.2. Soxhlet Extraction of Materials

After air drying, the leaves were subsequently dried and sterilized in an oven for 30 minutes in a 500 ml clean boiling flask at 110˚C. After that, it was placed in a desiccator and left to cool. 100 g of each sample were weighed and poured into the soxhlet thimble. To help filter the extract, the extraction thimble was lightly blocked with cotton wool, and the boiling flask was filled with 300 ml of ethanol. The soxhlet apparatus was put together and left to reflux for 4 hours at 600˚C. The thimble was carefully removed, and the extract was placed into a volumetric flask to cool. To separate the solvent (n-hexane) from the oil, the contents of the volumetric flask were transferred to a rotatory evaporator [5] .

2.3. Preparation of Stock Solution

One gram (1 g) of extract after soxhlet extraction was collected, weighed and placed in a test tube, along with 25 ml of ethanol. The test tube was placed on a hot plate at 600˚C for 90 minutes to respond. The reaction product in the test tube was transferred to a separatory funnel when the reaction period had passed. The tube was successfully cleansed with 20 ml ethanol, 10 ml cold water, 10 ml hot water, and 3 ml hexane, all of which were passed to the funnel. The extracts were mixed and rinsed three times with a 10 ml ethanol aqueous solution containing 10% v/v ethanol. The solvent was evaporated after the solution was dried with anhydrous sodium sulfate. The sample was dissolved in 1000 µ pyridine, 200 µl of which was transferred to a vial for GC-MS examination [6] .

2.4. The GC-MS Analysis

Agilent Technologies GC-7890A/MS-5975C a high-performance gas chromatography (GC) system manufactured by Agilent Technologies, based in Santa Clara, CA, USA equipped with a HP-5MS column, which is a type of high-performance liquid chromatography (HPLC) column that is commonly used in the analysis of volatile and semi-volatile organic compoundswere used to analyze bioactive chemicals from the various extracts. An electron ionization device using high energy electrons was used for spectroscopic detection by GC-MS (70 eV). The carrier gas was pure helium gas (99.995% purity) at a flow rate of 1 mL/min. The starting temperature was set at 50˚C - 150˚C, with a 3˚C/min increase rate and a 10-minute hold duration. Finally, the temperature was raised to 300˚C at a rate of 10˚C per minute. In a splitless mode, one microliter of the prepared 1 percent of the extracts diluted with corresponding solvents was injected. Based on the peak area obtained in the chromatogram, the relative quantity of chemical compounds contained in each of the extracts was represented as a percentage [5] .

2.5. Identification of Chemical Constituents

Based on GC retention time on HP-5MS column and spectra matching with computer software data of standards (Replib and Mainlab data of GC-MS systems), bioactive chemicals isolated from various extracts were identified. The National Institute of Standards and Technology (NIST) database, which contains over 62,000 patterns, was used to analyze the mass spectrum GC-MS. The spectra of the unknown components were compared to the spectrum of the known components in the NIST collection. The components in the test sample were identified by their name, molecular weight, and structure [7] .

2.6. Calculation of Retention Time

Retention time was calculated using the following principles below;

I^T was calculated using a quasi-linear equation for temperature programmed retention index:

I T = 100 ( t x − t n + n ) t n + 1 − t n

where I^T is the temperature-programmed retention index of the intriguing compound; t_n, t_n + 1, and t_x are the retention durations (in minutes) of the two standard n-alkanes with n and n + 1 carbons, respectively, and the compound of interest [7] .

3. Results

From the HP-5MS column and spectra matching with computer software data of standards (Replib and Mainlab data of GC-MS systems), the following results were obtained.

Table 1 presents the bioactive principles with their molecular formulas, molecular weight, retention time (RT), and percentage content (Percent).

The peak indicates the existence of bio-active chemicals, according to the analysis. GC-MS study of the Azadirachta indica leaf extract revealed thirty-five components. The bioactive components were identified and characterized and interpreted on mass spectrum GC-MS conducted using the database of National Institute Standard and Technology (NIST) which has more than 62,000 patterns.

Figures 1-12 show the mass spectrograms of twelve of the thirty-five compounds with the highest percentage of bioactive compounds, including; Methenamine (26.29%, RT 8.451); Hexadecanoic acid, methyl ester (14.54%, RT 15.790); Phenol (8.01%, RT 5.216); Dodecanoic acid, methyl ester (7.05%, RT 11.857); Methyl tetradecanoate (5.02%, RT 13.920); sec-Butylamine (4.88%, RT 6.638); 2-Propanone, 1-methoxy- (3.19%, RT 5.414); 10-Octadecenoic acid, methyl ester (3.12%, RT 17.283); Heptadecanoic acid, 16-methyl- (3.03%, RT 17.481); Phenol, 2,6-dimethoxy- (2.39%, RT 9.959); Phenol, 2-methoxy- (2.12%, RT 6.550); n-Hexadecanoic acid (2.08%, RT 16.152).

4. Discussion

Many bioactive elements found in plants are recognized to be physiologically active molecules that can be exploited in medication development. There is scientific proof in this study that the plants harvested with the advice and instructions of certain herbalists in the study region contain bioactive components that researchers have demonstrated to have bioactive actions against a variety of diseases and infections. The selection of these natural resources was motivated by herbalists' advice for using these materials to treat a variety of clinical and community illnesses, including upper respiratory infections, skin and urine infections, and sexually transmitted infections. All of these served as an encouragement to conduct a phytochemical study utilizing GC-MS as a method to look into bioactive chemicals.

By GC-MS analysis, 35 chemicals were found in Azadirachta indica leaf

Table 1 Phytocomponent identities of the hexane GC-MS chromatogram analyses of Azadirachta indica leaf

S/N	Parameters	Retention time	Molecular Formulae	Molecular weight (g/mol)	% composition
1.	Phenol	5.216	C₆H₆O	94.11	8.01
2.	2,4,6-cycloheptatrien-1-one	5.304	C₁₀H₁₂O₂	164.20	1.14
3.	2-Propanone, 1-methoxy-	5.414	C₄H₈O₂	88.11	3.19
4.	Methylvalerate	5.529	C₆H₈O₂	116.16	0.31
5.	3-methylcyclopentane-1,2-dione	5.718	C₆H₈O₂	112.13	1.54
6.	2-Pyrrolidinone	6.358	C₄H₇NO	85.11	0.65
7.	Phenol, 2-methoxy-	6.550	C₇H₈O₂	85.11	2.12
8.	sec-Butylamine	6.638	C₄H₈N₄	112.13	4.88
9.	1H-Imidazole, 1-methyl-4-nitro-	6.755	C₄H₅N₃O₂	127.10	0.81
10.	Octanoic acid, methyl ester	6.936	C₉H₁₈O₂	158.24	1.08
11.	4(1H)-Pyridone	7.136	C₅H₅NO	95.10	0.53
12.	3-Pyridinecarboxylic acid, 4-hydro	7.209	C₆H₅NO₃	139.11	1.50
13.	3(2H)-Isoxazolone, 4,5-dimethyl-	7.894	C₅H₇NO₂	113.11	0.68
14.	2-Hexenal, (E)-	7.977	C₆H₁₀O	98.14	0.78
15.	1,4:3,6-Dianhydro-.alpha.-d-glucop yranose	8.250	C₆H₈O₄	144.1253	1.12
16.	Methenamine	8.451	C₆H₁₂N₄	140.186	26.29
17.	1,2-Benzenediol, 3-methoxy-	8.961	C₇H₈O₃	140.1366	0.72
18.	Decanoic acid, methyl ester	9.545	C₁₁H₂₂O₂	186.29	1.77
19.	Phenol, 2,6-dimethoxy-	9.959	C₈H₁₀O₃	154.16	2.39
20.	Undecanoic acid, methyl ester	10.732	C₁₃H₂₆O₂	214.34	0.28
21.	3,5-Dimethoxy-4-hydroxytoluene	11.076	C₉H₁₂O	168.1898	0.38
22.	Dodecanoic acid, methyl ester	11.857	C₁₃H₂₆O₂	214.34	7.05
23.	5-tert-Butylpyrogallol	11.961	C₁₀H₁₄O₃	182.22	0.20
24.	Tridecanoic acid, methyl ester	12.911	C₁₆H₃₂O₂	256.42	0.41
25.	Methyl tetradecanoate	13.920	C₁₅H₃₀O₂	242.40	5.02
26.	Pentadecanoic acid, methyl ester	14.872	C₁₆H₃₂O₂	256.42	0.71
27.	(Z)-Methyl hexadec-11-enoate	15.606	C₁₇H₃₂O₂	268.4348	0.54
28.	Hexadecanoic acid, methyl ester	15.790	C₁₇H₃₄O₂	270.45	14.54
29.	n-Hexadecanoic acid	16.152	C₁₆H₃₂O₂	256.4	2..08
30.	Heptadecanoic acid, methyl ester	16.649	C₁₉H₃₈O₂	298.5	0.34
31.	10-Octadecenoic acid, methyl ester,	17.283	C₁₉H₃₆O₂	296.5	3.12
32.	11-Octadecenoic acid, methyl ester	17.331	C₁₉H₃₆O₂	296.5	1.10
33.	Heptadecanoic acid, 16-methyl-	17.481	C₁₉H₃₈O₂	298.5	3.03
34.	trans-13-Octadecenoic acid	17.638	C₁₈H₃₄O₂	282.5	0.25
35.	Diisooctyl phthalate	20.640	C₂₄H₃₈O₄	390.6	0.26

extract. The bioactive components were identified, characterized and interpreted on mass spectrum GC-MS conducted using the database of National Institute Standard and Technology (NIST) which has more than 62,000 patterns. Aside from that, the distinctive fragmentation patterns aided in the identification of a certain class of chemicals [8] . Table 1 indicate the bioactive principles with their molecular formulas, molecular weight, retention time (RT), and percentage content (percent) of the plant material. The following is the distribution of bioactive identities from the hexane GC-MS chromatogram of Azadirachta indica leaf. Methenamine, Hexadecanoic acid, methyl ester; Phenol; Dodecanoic acid, methyl ester; Methyl tetradecanoate; sec-Butylamine; 2-Propanone, 1-methoxy-; 10-Octadecenoic acid, methyl ester; Heptadecanoic acid, 16-methyl-; Phenol, 2,6-dimethoxy-; Phenol, 2-methoxy-; n-Hexadecanoic acid. This is consistent with the findings of Yuvarajan et al. [9] , who found several comparable phytochemicals and derivatives in low and high percentage proportions, respectively. Some of the phytochemical with high percentage composition determined in this study is in agreement with the study. They include; phenol, [10] , 2-Propanone, 1-methoxy- [11] , Phenol, 2-methoxy [12] , Hexadecanoic acid, methyl ester [13] , Dodecanoic acid, methyl ester, Hexadecanoic acid, methyl ester, and 10-Octadecenoic acid, methyl ester have been described by Bolade et al. [14] . The current study reviewed that bioactive compounds such as sec-Butylamine and Methenamine with elevated composition obtained in the extracts examined in this study had not previously been determined by other researchers. This could be due to the plants’ biosynthetic pathway, which is heavily influenced by many ecological factors such as temperature, humidity, soil type and nutrients, and atmospheric pressure. Furthermore, the catalytic enzymes may differ. sec-Butylamine and Methenamine has antibacterial properties [15] . There is a significant difference in the outcomes of bioactive chemicals determined from the current investigation.

Zih-Rou et al. [16] ; Shi et al. [17] ; Nayak et al. [18] ; Bartnik et al. [19] ; Yong et al. [20] ; Madrona et al. [21] ; Pant et al. [22] ; Saloni et al. [23] ; Meechaona et al. [24] ; Adsul et al. [25] ; Oon et al. [26] ; Kasture et al. [27] and Scortichini et al. [28] explained the medicinal important of Azadirachta indica (Neem) as follows: anti-inflammatory, hepatoprotective effect, wound healing effect, antidiabetic activity, antinephrotoxicity effect, neuroprotective effects, antimicrobial effect, immunomodulatory and growth promoting effect and that A. indica mouth rinse is equally effective in reducing periodontal indices which justifies the result obtained in the present study. All of these phytochemicals play a significant role in the formulation of various medicinal products. As antioxidants, phenol, sec-Butylamine, methyl tetradecanoate, hexadecanoic acid, and methyl ester have been employed [15] [29] [30] [31] .

5. Conclusion

This research has revealed that Azadirachta indica possess vital bioactive compounds that have been found to have a wide range of biological properties. The structure of active chemicals that might be exploited in drug production was also shown. Our findings also support the traditional use of Azadirachta indica, which contains a variety of bioactive components as antimicrobial agents. There is therefore need for a single fractionalization and separation of active compounds for a single slope antibacterial activity, which can aid in the utilization of these compounds in manufacture of pharmaceutical products.

Ethics Approval

The plants were identified, validated and recognized by a Plant Taxonomist at Imo State University, Department of Crop Science, based on their taxonomic classification and further deposited in the herbarium Department of Crop Science, Imo State University Owerri with the voucher number DPS/IMSU/0621.

Consent for Publication

The authors have declared that neither the manuscript nor any parts of its content are currently under consideration or published in another journal and all authors have approved the manuscript and agree with its submission and publication in Bulletin of the National Research Centre.

Availability of Data and Material

Data is contained within the article.

Funding

This research was self-funded as a postgraduate thesis and did not receive any external funding.

Author Contributions

IFC, OSI, NRI and CIC, designed the work. OSI, NRI and CIC supervised the study. IFC and AII conducted the sampling and survey. IFC analyzed the data. IFC and CIC wrote the paper. All authors read and approved the final manuscript.

Acknowledgments

We are grateful to Microbiology Unit of Federal Medical (FMC) Center Owerri and the plant taxonomist who identified the plant.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Ihenetu, F.C., Okorondu, S.I., Chikwendu, C.I., Nwabueze, R.N. and I.I. Anekwe (2024) Identification of Sec-Butylamine and Methenamine and Other Bioactive Compounds from Edible Azadirachta indica Methanolic Extracts Using GC-MS. Open Access Library Journal, 11: e9750. https://doi.org/10.4236/oalib.1109750

List of Abbreviations

GC-MS: Gas Chromatography-Mass Spectrometry

BCVs: Branch Chain Volatiles

GC-FID: Gas Chromatography with Flame Ionization Detector

NIST: The National Institute of Standards and Technology

References1

Haznagy-Radnal, E., Czigle, S. and Mathe, I. (2007) TLC and GC Analysis of the Essential Oils of Stachys Species. Journal of Planar Chromatography, 20, 189-196.https://doi.org/10.1556/JPC.20.2007.3.5

Wang, Y., Chang, L., Zhao, X., Meng, X. and Liu, Y. (2012) Gas Chromatography-Mass Spectrometry Analysis on Compounds in Volatile Oils Extracted from Yuan Zhi (Radix polygalae) and Shi Chang Pu (Acorus tatarinowii) by Supercritical CO2. Journal of Traditional Chinese Medicine, 32, 459-464.https://doi.org/10.1016/S0254-6272(13)60055-2

Syeda, R., Holden, M.A., Hwang, W.L. and Bayley, H. (2008) Screening Blockers against a Potassium Channel with a Droplet Interface Bilayer Array. Journal of the American Chemical Society, 130, 15543-15548. https://doi.org/10.1021/ja804968g

Mongkholkhajornsilp, D., Douglas, S., Douglas, P.L., Elkamel, A., Teppaitoon, W. and Pongamphai, S. (2005) Supercritical CO2 Extraction of Nimbin from Neem Seeds—A Modelling Study. Journal of Food Engineering, 71, 331-340.https://doi.org/10.1016/j.jfoodeng.2004.08.007

Mass Spectrometry Data Centre (1974) Eight Peak Index of Mass Spectra: The Eight Most Abundant Ions in 31,101 Mass Spectra, Indexed by Molecular Weight, Elemental Composition and Most Abundant Ions (4 Volume Set). 2nd Edition, Mass Spectrometry Data Centre, Aldermaston.

Zhang, C., Qi, M., Shao, Q., Zhou, S. and Fu, R. (2007) Analysis of the Volatile Compounds in Ligusticum chuanxiong Hort. Using HS-SPME-GC-MS. Journal of Pharmaceutical and Biomedical Analysis, 44, 464-470.https://doi.org/10.1016/j.jpba.2007.01.024

Weernink, A., Severin, W.P.J., Tjernberg, I. and Dijkshoorn, L. (1995) Pillows, an Unexpected Source of Acinetobacter. Journal of Hospital Infection, 29, 189-199.https://doi.org/10.1016/0195-6701(95)90328-3

Anees, T.P., Hisham, M., Sekhar, M., Madhu, M. and Deepa, T. (2010) International Market Scenario of Traditional Indian Herbal Drugs: India Declining. International Journal of Green Pharmacy, 3, 184-190.https://doi.org/10.4103/0973-8258.56271

Yuvarajan, D., Lokesh, C. and Balaji, P. (2016) Analysis on Influence of Varying Compression Ratio in Biofuel. Applied Mechanics and Materials, 852, 734-738.https://doi.org/10.4028/www.scientific.net/AMM.852.734

Patil, S.M., Shirahatti, P.S., V B, C.K., Ramu, R. and M N, N.P. (2021) Azadirachta indica A. Juss (neem) as a Contraceptive: An Evidence-Based Review on Its Pharmacological Efficiency. Phytomedicine, 88, Article 153596.https://doi.org/10.1016/j.phymed.2021.153596

Rafiqul Islam, A.T.M., Hasan, M., Islam, T. and Tanaka, N. (2021) Ethnobotanical Study of Plants Used by the Munda Ethnic Group Living around the Sundarbans, the World’s Largest Mangrove Forest in Southwestern Bangladesh. Journal of Ethnopharmacology, 285, Article 114853. https://doi.org/10.1016/j.jep.2021.114853

Swapna, S.R., Ramalakshmi, K. and Udaya, S.K. (2018) Characterization of Neem (Azadirachta indica A. Juss) Seed Volatile Compounds Obtained by Supercritical Carbon Dioxide Process. Journal of Food Science and Technology, 55, 1444-1454.https://doi.org/10.1007/s13197-018-3060-y

Ismaila, S.S., Sani, Y., Sani, A.A., Yakasai, S.M., Momoh, H. and Sa’adatu, E.M. (2022) Determination of Fatty Acids and Physicochemical Properties of Neem (Azadrachta indica L) Seed Oil Extracts. Dutse Journal of Pure and Applied Sciences, 8, 149-160. https://doi.org/10.4314/dujopas.v8i1a.16

Bolade, O.P., Williams, A.B. and Benson, N.U. (2021) Dataset on Analytical Characterization of Bioactive Components from Azadirachta indica, Canna indica, Magnifera indica and Moringa oleifera Leaf Extracts and Their Applications in Nanoparticles Biosynthesis. Data in Brief, 38, Article 107407.https://doi.org/10.1016/j.dib.2021.107407

Opdyke, D.L.J. and Letizia, C. (1982) Fragrance Raw Materials Monographs. Food and Chemical Toxicology, 20, 637-852.https://doi.org/10.1016/S0015-6264(82)80217-4

Huang, Z.-R., Lin, Y.-K. and Fang, J.-Y. (2009) Biological and Pharmacological Activities of Squalene and Related Compounds: Potential Uses in Cosmetic Dermatology. Molecules, 14, 540-554. https://doi.org/10.3390/molecules14010540

Shi, H.L., Noguchi, N. and Niki, N. (1999) Comparative Study on Dynamics of Antioxidative Action of α-Tocopheryl Hydroquinone, Ubiquinol and α-Tocopherol, against Lipid Peroxidation. Free Radical Biology and Medicine, 27, 334-346.https://doi.org/10.1016/S0891-5849(99)00053-2

Nayak, M.G., George, A., Vidyasagar, M.S., Stanley, M., Nayak, S., Nayak, B.S., Shashidhara, Y.N. and Kamath, A. (2017) Quality of Life among Cancer Patients. Indian Journal of Palliative Care, 23, 445-450.https://doi.org/10.4103/IJPC.IJPC_82_17

Bartnik, F.G., Reddy, A.K., Klecak, G., Zimmermann, V., Hostynek, J.J. and Kunstler, K. (1987) Percutaneous Absorption, Metabolism, and Hemolytic Activity of n-Butoxyethanol. Toxicological Sciences, 8, 59-70.https://doi.org/10.1093/toxsci/8.1.59

Yong, S.T., Mardhati, M., Farahiyah, I.J., Noraini, S. and Wong, H.K. (2018) Replacement of Fishmeal in Feather Meal-based Diet and Its Effects on Tilapia Growth Performance and on Water Quality Parameters. Journal of Tropical Agriculture and Food Science, 46, 47-55.

Madrona, A., Pereira-Caro, G., Mateos, R., Rodríguez, G., Trujillo, M., Fernández-Bolaños, J. and Espartero, J.L. (2009) Synthesis of Hydroxytyrosyl Alkyl Ethers from Olive Oil Waste Waters. Molecules, 14, 1762-1772.https://doi.org/10.3390/molecules14051762

Pant, B. (2013) Medicinal Orchids and their Uses: Tissue Culture a Potential Alternative for Conservation. African Journal of Plant Science, 7, 448-467.https://doi.org/10.5897/AJPS2013.1031

Saloni, K., Patel, J., Barve, K. and Londhe, V. (2019) Herbal Nano-Formulations for Topical Delivery. Journal of Herbal Medicine, 20, Article 100300.https://doi.org/10.1016/j.hermed.2019.100300

Meechaona, R., Sengpracha, W., Banditpuritat, J., Kawaree, R. and Phutdhawong, W. (2007) Fatty Acid Content and Antioxidant Activity of Thai bananas. Maejo International Journal of Science and Technology, 1, 222-228.

Adsul, M.G., Bastawde, K.B., Anjani, V. and Digambar, G. (2007) Strain Improvement of Penicillium janthinellum NCIM 1171 for Increased Cellulase Production. Bioresource Technology, 98, 1467-1473.https://doi.org/10.1016/j.biortech.2006.02.036

Oon, S.F., Nallappan, M., Tee, T.T., Shohaimi, S., Kasim, N.K., Sa’ariwijaya, M.S.F. and Cheah, Y.H. (2015) Xanthorrhizol: A Review of Its Pharmacological Activities and Anticancer Properties. Cancer Cell International, 15, Article No. 100.https://doi.org/10.1186/s12935-015-0255-4

Kasture, S., Pontis, S., Pinna, A., Schintu, N., Spina, L., Longoni, R., Simola, N., Ballero, M. and Morelli, M. (2009) Assessment of Symptomatic and Neuroprotective Efficacy of Mucuna pruriens Seed Extract in Rodent Model of Parkinson’s Disease. Neurotoxicity Research, 15, 111-122.https://doi.org/10.1007/s12640-009-9011-7

Scortichini, M. and Rossi, P.M. (1991) Preliminary in vitro Evaluation of Antimicrobial Activity of Terpenes and Terpenoids towards Erwinia amylovora (Burrill). Journal of Applied Bacteriology, 71, 109-112.https://doi.org/10.1111/j.1365-2672.1991.tb02963.x

Becker, H., Scher, J.M., Speakman, J.B. and Zapp, J. (2005) Bioactivity Guided Isolation of Antimicrobial Compounds from Lythrum salicaria. Fitoterapia, 76, 580-584.https://doi.org/10.1016/j.fitote.2005.04.011

Pejin, B., Kojić, V. and Bogdanovic, G. (2014) An Insight into the Cytotoxic Activity of Phytol at in vitro Conditions. Natural Product Research, 28, 2053-2056.https://doi.org/10.1080/14786419.2014.921686

Villas-Boas, S.G., Mas, S., Akesson, M., Smedsgaard, J. and Nielsen, J. (2005) Mass Spectrometry in Metabolome Analysis. Mass Spectrometry Reviews, 24, 613-646.https://doi.org/10.1002/mas.20032