INTRODUCTION:

Phytoconstituents ('phyto-' meaning 'plant' in Greek) or phytochemicals are bioactive ingredients found in numerous plant species.^1,2 Naturally, these chemicals play a crucial function in protecting plants from microbial or insect-caused infection or attack.³^,⁴

The production of medicinal plants based on ethnomedicine for a variety of diseases, such as tuberculosis, cancer, and inflammatory disorders such as diabetes, is a popular community preference.^5,6 If handled correctly, the usage of medicinal herbs is often safer in terms of side effects and toxicity compared to contemporary pharmaceuticals.^2,7,8 The chemical compositions of plants utilized in phytomedicine are complicated and comprise numerous components. The age of the plant, geographical origin (climatic differences), growing conditions, genetic composition of plants, varieties, plant species, and portions of plants chosen for processing all influence the quality of the elements and the content of the active components in herbal medicines.⁹^,^10,11

The plants of the genus Ocimum, which are members of the family Lamiaceae (Ordo Lamiales) and are known as tulsi, are extensively distributed in tropical, subtropical, and warm climates all over the world.^12,13 It is classified as types of herbal plants that have been demonstrated to have medicinal benefits.^14,15 This plant is reported to produce an oil containing a variety of aromatic chemicals. Secondary metabolites (essential oils) of the genus Ocimum are recommended for the treatment of malaria, bronchitis, diarrhea, dysentery, etc. due to their anticancer, antioxidant, antifungal, and anti-inflammatory effects.^16,17 Ocimum species have been found to contain significant metabolites such as linalool, linalyl, geraniol, citral, camphor, eugenol, methyleugenol, methyl chavicol, methyl cinnamate, thymol, safrole, taxol, urosolic acid, etc. In the pharmaceutical, perfume, and cosmetics industries, these metabolites are highly lucrative.^15,18,19

a b

Figure 1. a. Khrisna Tulsi, b. Rama tulsi

As in India, various types of Tulsi plants grow in Indonesia, including on the island of Bali, with Tulsi Rama and Tulsi Krishna being the most prevalent and widely utilized as herbal medicine or tea. The stems, leaves, and flowers of the two types of tulsi are distinct in terms of color. These morphological differences may result in distinct secondary metabolite compositions. Consequently, the profile of secondary metabolites from Tulsi Rama was characterized in this study, and future research will do the same for Tulsi Krishna.

In India, plants of the genus Ocimum are commonly utilized in traditional medicine, however in Bali, tulsi is not frequently used as an alternative to herbal medicine, and there is a dearth of research describing its chemical constituents and secondary metabolites. Geographical and genetic variables of the plant itself influence the content of plant metabolites, allowing for variances in metabolites and the associated pharmacological reactions.¹⁵ Therefore, it is necessary to standardize the extract as a raw material for the phytopharmaceutical in order to be accounted for medically.In this study, polar 95% ethanol and non-polar chloroform solvents were used in the maceration procedure. The purpose of using these two solvents is to characterize all types of secondary metabolites in the tulsi plant, polar and non-polar alike.

MATERIALS AND METHODS:

Sample Collection and Extraction:

The sample utilized was collected at an altitude of 425 masl in the city of Gianyar. The organs used as samples were stems and leaves. The samples were dried in an oven at a temperature between 50 and 60 degrees Celsius to achieve a constant dry mass. To be able to characterize all sorts of secondary metabolites with differing polarities, two solvents with varying polarity levels are utilized. The sample was pulverized using a blender and macerated with 95% ethanol and chloroform at a ratio of 3:1 (sample powder: ethanol/chloroform) in a sealed aluminum foil container (vacuum sealed aluminum). Each 24 hours of the 46-hour maceration process were filtered, and the sample was soaked in new 95% etanol and chloroform. The extract was then filtered using filter paper to obtain the filtrate, which was then evaporated in the dark using an evaporator to produce a precipitate. The evaporation of the precipitate results in the production of crude extract (ethanolic and chloroform extract).

Secondary Metabolites Characteristics (GC-MS test):

In addition, the ethanol and chloroform extracts will be phytochemically evaluated with GC-MS Agilent 7890B and MSD 5977 A. The GC-MS was injected with a 1 μL sample.

RESULTS:

In the ethanolic extract of tulsi, six types of secondary metabolites with GC-MS peak quality above 60% were found. With a percentage of covered area of 44.12%, Cyclohexane,1-ethenyl-1-methyl-2,4-bis(1-methylethyl) had the highest concentration, followed by Eugenol (17.20%), Germacrene D (10.24%), Caryophyllene (10.23).%), N-Desmethyl tapentadol (3.56%), and Alpha-Copaene (1.92%). In addition, retention time, molecular formula, molecular weight, and boiling point information for each of these secondary metabolites is presented in table 1. Figure 1 depicts the GC-MS chromatogram of the ethanolic extract of tulsi (Ocimum tenuiflorum).

Table 1: Chemical constituents (secondary metabolites) found in tulsi ethanolic extract (Ocimum tenuiflorum)

Constituents	Type of secondary metabolites	Retention Time (RT) (Minutes)	Molecular formula	Molecular Weight (g/mol)	Peak area	Boiling point
Eugenol (4-allyl-2-methoxyphenol)	Terpenoid-alkaloids	7.153	C₁₀H₁₂O₂	164.2	17.20	254^oC
Alfa-Copaene	Sesquiterpenes	7.335	C₅H₂₄	204.35	1.96	248.5^oC
Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)	Benzene	7.426	C₁₅H₂₄	204.35	44.12	80.75^oC
Caryophyllene	Sesquiterpenes	7.665	C₁₅H₂₄	204.36	10.23	245.3°C
N-Desmethyl tapentadol	Alkylbenzene	9.038	C₁₃H₂₁NO	207.31	3.56	123^oC
Germacrene D	Sesquiterpenes	8.112	C₁₅H₂₄	204.35	10.24	236.4°C

Table 2: Chemical constituents (secondary metabolites) discovered in chloroform tulsi extract (Ocimum tenuiflorum)

Constituents	Type of secondary metabolites	Retention Time (RT) (Minutes)	Molecular formula	Molecular Weight (g/mol)	Peak area	Boiling point
3-allyl-6-methoxyphenol	Phenolic	7.152	C₁₂H₁₄O₃	206.24	27.82	127^oC
Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)	Benzene	7.379	C₁₅H₂₄	204.35	30.81	80.75^oC
Copaene	Sesquiterpenes	7.335	C₅H₂₄	204.35	1.42	248.5^oC
Caryophyllene	Sesquiterpenes	7.665	C₁₅H₂₄	204.36	6.55	245.3°C
Humulene	Sesquiterpenes	7.911	C₁₅H₂₄	204.35	0.56	106°C
Germacrene D	Sesquiterpenes	8.112	C₁₅H₂₄	204.35	7.94	236.4°C
Naphthalene	Aromatic compounds	8.404	C₁₀H₈	128.1705	1.25	218 °C
Caryophyllene oxide	Sesquiterpenes	9.038	C15H24O	220.3505	2.61	279.6°C
9,12,15-Octadecatrienoic acid	Methylester, Polyunsaturated fatty acid	16.698	C₁₈H₃₀O₂	278.43	4.48	443.4°C
N-Desmethyl tapentadol	Alkylbenzene	8.704	C₁₃H₂₁NO	207.31	0.50	123^oC
Dibutyl phthalate	phthalate ester	13.243	C₆H₄(CO₂C₄H₉)₂	278.3	4.30	340°C

Table 3: Advantages of secondary metabolites found in ethanol and chloroform extract of tulsi (Ocimum tenuiflorum)

Constituents	Chemical Structure	Biological potential
Eugenol (4-allyl-2-methoxyphenol)		Biological activity as an antifungal, antibacterial, antimicrobial, anti-inflammatory, analgesic, antioxidant, anticancer, and antiparasitic agent.^20–22
Alfa-Copaene		Antibacterial efficacy against several Streptococcus bacterium, Actinomyces naeslundii, Bacteroides fragilis, Prevotella nigrescens²³andXanthomonas oryzae²⁴
Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)		Strong scent, flavor, and spice level. Additionally, it has been demonstrated to suppress various types of harmful microorganisms.²⁵^,21
Caryophyllene		Numerous in vitro and in vivo investigations demonstrate that caryophyllene has an anti-inflammatory activity. ⁷ In addition, it has anticonvulsant, analgesic, myorelaxing, sedative, and antidepressant properties. ^{8– 10,}
N-Desmethyl tapentadol		possesses qualities suitable for development as an analgesic medication ^26,27
Germacrene D		It exhibits antiparasitic, anti-inflammatory, antibacterial, and anti-worm activities and has pesticide potential.^28,29
3-allyl-6-methoxyphenol		Contains antibacterial, antifungal, and antioxidant qualities, as well as pesticide potential. ^30,31
Humulene		In addition to possessing antioxidant, anti-inflammatory, and antibacterial characteristics, it has the potential to be developed as a larvicide.^32–34
9,12,15-Octadecatrienoic acid		It has been shown to lessen the risk of fatal ischemia in patients with heart disease due to its antioxidant and anti-inflammatory characteristics. Additionally, it possesses anticancer properties.^35,36
Dibutyl phthalate		In in addition to their antibacterial and antifungal characteristics, these chemicals also possess hazardous qualities, particularly with regard to reproduction.³⁷

DISCUSSION:

In this investigation, two polarity-distinct types of organic solvents were utilized. In order to bind more polar/semipolar molecules, 95 percent ethanol is employed in the maceration process. While chloroform is used to bind the lipophilic group of secondary metabolites, chloroform is utilized to bind the hydrophilic group of secondary metabolites. Using either ethanol or chloroform as solvents, many types of secondary metabolites were discovered in tulsi extract.

Ocimum tenuiflorum tulsi extract contains antistress, antiseptic, analgesic, anti-inflammatory, antimicrobial, immunomodulatory, hypoglycemic, hypotensive, cardioprotective, and antioxidant activities, according to various research.^9,38,39 The secondary metabolites discovered in the ethanol and chloroform extracts of the tulsi plant have diverse biological activities, as detailed in Table 3. Eugenol (4-allyl-2-methoxyphenol) is one of the secondary metabolites that has the potential to be developed as a therapeutic agent due to its antiseptic, analgesic, anti-inflammatory, antibacterial, immunomodulatory, hypoglycemic, hypotensive, cardioprotective, and antioxidant properties.^20–22

In a number of investigations, cyclohexane and its derivative 1-ethenyl-1-methyl-2,4-bis(1-methylethyl) inhibited the development of harmful bacteria, including S. aureus, S. aeruginosa, and E. coli.²¹ Moreover, this secondary metabolite was found in significant concentrations in both the ethanol and chloroform extracts of the tulsi plant. It demonstrates that both types of extracts have a tremendous amount of potential as natural antibacterials derived from herbal plants. In addition, the Caryophyllene chemical, which is included in the Sesquiterpenes compound group, has been demonstrated in vitro and in vivo to have anti-inflammatory properties by various research.^40–43This substance not only has anti-inflammatory properties, but it can also impede the growth of Streptococcus bacteria. 34 This secondary metabolite molecule exhibits antidepressant properties; hence it has the potential to be developed as a sedative ingredient.²⁸

Moreover, a number of the compounds discovered in these two types of tulsi plant extracts have intriguing biological features, including the potential to be developed as larvicides and natural insecticides, especially against the Aedes mosquito, which is a vector for a number of dangerous infections.²⁸ Multiple studies of the Germacrene D, 3-allyl-6-methoxyphenol, and Humulene groups have revealed that, although being toxic to Aedes larvae and adult mosquitoes, these three compounds are not dangerous to humans at particular concentrations (proven by in vitro and in vivo tests). Consequently, the extract of the tulsi plant has the potential to be developed as both a larvicide and a natural insecticide, despite the fact that multiple studies have proven that mosquitoes possess a resistance mechanism to chemical insecticides.²³ Consequently, the extract of the tulsi plant has tremendous potential to be developed as a larvicide as well as a natural insecticide, despite the fact that numerous studies have demonstrated that mosquitoes exhibit a resistance mechanism to chemical insecticides.^19-22

CONCLUSION:

Two different solvents, ethanol and chloroform, were used to extract secondary metabolites from the tulsi plant. The majority of these secondary metabolites exhibit antipathogenic properties, including antifungal and antibacterial activities. The cyclohexane molecule with the highest concentration in both ethanol extract and chloroform possesses antibacterial properties and has the potential to be developed into various antipathogens, such as antifungals. In addition, the Caryophyllene component, which belongs to the Sesquiterpenes compound family, has been demonstrated by multiple studies to have anti-inflammatory properties. Various secondary metabolites in these two forms of tulsi extract have the potential to be developed as therapeutic agents due to their antiseptic, analgesic, anti-inflammatory, antibacterial, immunomodulatory, hypoglycemic, hypotensive, cardioprotective, and antioxidant activities. There are variations in the types and amounts of secondary metabolites present in each extract, resulting in distinct biological effects/capabilities that require additional study.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We are grateful to the Research Institute of Universitas Warmadewa for providing the grant to finance this study, as well as to all academic and laboratory employees for their support and assistance throughout the process of this study.

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Received on 23.10.2022 Modified on 14.12.2022

Research J. Pharm. and Tech 2023; 16(9):4377-4383.

DOI: 10.52711/0974-360X.2023.00716