INTRODUCTION:

Zanthoxylum armatum DC. belongs to family Rutaceae is a pharmaceutically important plant that has been claimed to be effective against various diseases in the folk medicinal system of Indian subcontinent^1,2. The genus Zanthoxylum is economically important due to its nutritional, industrial and therapeutic values. Z. armatum is a short tree or a shrub that grows under dry conditions³. It is known as Timur also called Nepali pepper in Nepal, Toothache plant or Prickly ash in English, Darmar orTejfal in Hindi, Tejovati orDhiva in Sanskrit. In natural habitat, it grows up to 6 m in height with dense foliage and armed branched flattened prickles.

Leaves are compound, 4 to 20cm long, imparipinnate, rachis winged, serrate with gland dots and aromatic, containing a flavour like lime and mint^4,5,6. The dried fruit also contain an aroma that is present in brown fruit wall (pericarp-shell), it may be able to develop numbing or anaesthetic feeling on the tongue, seeds are solitary, globose, shining and have bitter taste⁷.

The fruit and seeds are used as an aromatic tonic to treat fever, indigestion, and to get rid of round worms. Moreover, it can be used as a flavouring agent^8,9. Timur is not only used as flavouring in cooking but also its seed oil and crushed seeds are added to cereal seeds and legumes to protect them against damages caused by stored grain pests¹⁰, because the genus is a rich source of various chemicals such as amides, alkaloids, flavonoids, lignans, sterols and terpenes etc. Species of genus Zanthoxylum are of economic importance as source of edible fruits, essential oils, wood, ornamentals and raw materials for industries, medicinal plants and culinary applications etc.^11,12.

It is a well-established phenomenon that the microbial infections are the most common cause of deaths worldwide especially in developing and underprivileged countries. However, there are plethora of antibiotic drugs available in the market with potent efficacy but the side effect of these drugs limits their usages. For example, mayo clinic in the U.S.A. has enlisted the possible toxic effect of ciprofloxacin on their website which include burning, crawling, itching, numbness, prickling, "pins and needles", or tingling feelings, chest pain, discomfort, tightness, or heaviness and many more¹³. Moreover, the multiple drug resistance has become very common nowadays due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious disease. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plant as antimicrobials of plant origin have enormous therapeutic potential^14,15,16.

The plant essential oils are a good source of several bioactive compounds that possess antioxidative and antimicrobial properties^17,18. Z. armatum essential oil, commonly known as Timur oil is highly valued for commercial purpose. Several studies have been carried out on the essential oil composition of the fruits, seeds, and leaves of Z. armatum which reveal that the main constituents of the oil are linalool and limonene^19,20,21. In addition, fruits essential oil showed antiseptic, disinfectant and deodorant properties which might be ascribed to the presence of linalool and limonene²². Antimicrobial property of essential oils of medicinal plants havealsobeen reviewed²³, and are being developed for the treatment of infectious diseases ^24,25. Therefore, we in the present study explored the chemical profiling of the essential oil of the Zanthoxylum armatum seedsand evaluated its antibacterial efficacy against common human pathogens.

MATERIALS AND METHOD:

Materials:

Zanthoxylum armatum seeds were collected from Munsyari region, Pithoragarh, Uttarkahand, India. Nutrient Agar (NA) and Nutrient Broth (NB) were procured from Himedia, India. DMSO and methanol were procured from Sigma-Aldrich, USA. Standard drug gentamycin was procured from Himedia, India.

Test Organisms:

The microorganisms were procured from Microbial Type Culture Collection and Gene Bank (MTCC) Chandigarh. Escherichia coli (MTCC 40), Bacillus cereus (MTCC 6840), Staphylococcus aureus (MTCC 7443), Pseudomonas aeruginosa (MTCC 1668) were selected for study. The cultures of bacteria were maintained in their appropriate agar slants at 4°C throughout the study and used as stock cultures.

Extraction of essential oil from Z. armatum seeds:

In the laboratory, freshly collected seeds were crushed and hydrodistilated for 5 hours using the Clevenger apparatus. The essential oils obtained were immediately dried over anhydrous sodium sulphate in a glass vial. The samples were then kept at a low temperature (0- 4°C) until they were further analysed.

Gas- Chromatography -Mass Spectrometry (GC-MS) analysis of Timur essential oil:

GC-MS analysis of the oil was performed with a Perkin Elmer Claurs 500 gas chromatography equipped with a split/splitless injector (split ratio 50:1) data handling system. The column was Rtx5 capillary column (60 m × 0.32mm, 0.25μm film thickness). Helium (He) was the carrier gas at a flow rate 1.0mL/min. The GC was interfaced with (Perkin Elmer Clarus 500) mass detector operating in the EI positive mode. The mass spectra were generally recorded over m/z 40-500 amu that revealed the total ion current (TIC) chromatograms. Temperature program was used as the same as described above for GC analysis. The temperature of the injector, transfer line and ion source were maintained at 210°C, 210°C and 200°C respectively. Identification of the components was done on the basis of retention time, retention indices, determined with reference to homologous series of n-alkanes (C8 -C24 Sigma-Aldrich) under identical experimental conditions, co-injection with authentic standard compounds, mass spectra with library provided by instrument software (NIST/Wiley) and by comparing their mass spectra with those reported in literature ²⁶. Quantification of each compound was performed on the basis of their GC peak area, using the normalization procedure without using correction factors.

Preparation of inoculum:

To prepare the culture inoculumthree or four isolated colonies were picked and dissolved in the 2 ml nutrient broth and incubated till the growth in the broth was equivalent with Mac-Farland standard (OD-0.5) for the antimicrobial susceptibility test as recommended by WHO²⁷.

Zone of inhibition assay for Timur seeds essential oil:

The antibacterial activity of Z. armatum seeds oil was determined using the agar well diffusion method. The inoculum of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus,was done as mentioned above. The Nutrient agar plates were prepared and allowed to dry under sterile environment thereafter, a 0.1 ml of diluted inoculumwas spread over the agar plate with a sterile L shape spreader to achieve confluent growth. Using a sterile Cork borer, 6mm diameter wells were cut from the agar and 100µl of the essential oil was then filled into the wells. Gentamycin (10µg/well) was used as standard for E. coli, S. aureus, P. aeruginosa and B. cereus. The plates were then incubated for 24 hrs at 37°C. After the incubation period was completed the zone of inhibition was measured by using a scale and the data were expressed in mm.

Minimum inhibitory concentration (MIC) of Zanthoxylumarmatum seeds oil:

Determination of minimum inhibitory concentration (MIC) was done by broth dilution technique against the selected bacteria. Briefly, stock solution of essential oils was diluted in 1% methanol and further dilutions were madein nutrient broth to obtain a concentration of 1, 5, 10 and 20mg/ml. Each test tube of different concentrations was inoculated with microbial suspension, equivalent to 0.5 McFarland standards and incubated at 37°C for 24 hrs²⁸.The lowest concentration at which no bacterial growth was observed corresponded to the minimum inhibitory concentration (MIC)²⁹.

RESULTS AND DISCUSSION:

Z. armatum plant is well known for its tremendous medicinal uses in tradition settings for example, its fruit and seed (Fig. 1) has been used as an aromatic tonic for fever and dyspepsia³⁰. Additionally, the fruit, branches and thorns of the Timur plant are used to cure toothache, however, the bark part has been shown to be effective against the gum bleeding². Moreover, the essential oil of Timur is found to be effective in treatment of gastrointestinal and respiratory disorders³¹. These properties of various part of Timur plant signifies its medicinal value, nonetheless, a number of the studies has been done on the extracts of the various plants and less work has been explored on its seed essential oil. Therefore, we in the present study aimed to explore chemical composition and the antibacterial property of Timur seed oil located in Munsyari region Pithoragarh, Uttarakhand, India.

The chemical composition of plant decides the fate of its biological activity and this is the reason why we have carried out the GC-MS analysis of collected Timur seeds essential oil. The results of the study suggested that the linalool (32.53%) and limonene (27.15%) were the major components in the oil along with trans-Methyl cinnamate (19.32%) and β-Myrcene (6.10%) as depicted in Table 1 and Fig. 2. The linalool compound has been reported to have strong antibacterial efficacy and it acts on the membrane potential of the bacteria along with the nucleic acid damage against P. fluorescens. Nonetheless, linalool also dysregulates the metabolic activity and it decreases the respiratory chain dehydrogenase activity of P. fluorescens^32,33. Similarly, limonene anothermajor compound found in Timur seed oil also possesses significant antibacterial and antifungal activity against variety of pathogens. It is evident that limonene affects the membrane integrity of the both Gram positive and Gram negative bacterial resulting in to the cellular leakage³⁴.

Figure. 1. Dried seeds of Zanthoxylum armatum (Timur) plant.

Table 1. The chemical composition of Z. armatum seeds essential oil analysed by GC-MS.

S. No	Component	RT	RI^Lit.	RI^Exp.	Area (%)
1	α-Thujene	8.80	924	929	0.28
2	α-Pinene	9.06	932	937	0.41
3	Sabinene	10.50	969	974	1.07
4	β-Pinene	10.65	974	979	0.29
5	β-Myrcene	11.12	988	991	6.10
6	α-Phellandrene	11.70	1002	1005	0.59
7	α- Terpinene	12.19	1014	1017	0.73
8	p-Cymene	12.52	1020	1025	0.42
9	Limonene	12.71	1024	1018	27.15
10	γ- Terpinene	13.96	1054	1060	1.03
11	trans-Linalool oxide	14.56	1084	1086	0.58
12	α- Terpinolene	15.25	1086	1088	1.33
13	Linalool	15.76	1095	1099	32.53
14	Terpinen-4-ol	19.19	1174	1177	1.37
15	α- Terpineol	19.77	1186	1189	0.45
16	trans-Methyl cinnamate	28.15	1376	1379	19.32
17	Caryophyllene	29.68	1417	1419	0.28
18	α- Humulene	31.06	1452	1454	0.04
Monoterpene Hydrocarbons					39.40
Oxygenated Monoterpenes					54.25
Sesquiterpene Hydrocarbons					0.32
Total (%)					93.99

RI^{Lit. -}Retention indices (Literature); RI^Exp^.- Retention indices (experiment)

On the other hand, trans-Methyl cinnamate which was also evident in GC-MS analysis (19.32%) has also been well studied for its antimicrobial properties. For example, a study carried out by Huang et al. demonstrated that trans-Methyl cinnamate was effective against E. coli, B. subtilis and S. aureus. Also, the study revealed that it has anti-fungal activity as well against C. albicans³⁵. Moreover, β-Myrcenean abundant monoterpene found in Timur oil in our study (6.10%) has also been reported to have antibacterial activity against both Gram positive (S. aureus, M. luteus, B. cereus, and E. faecalis) and Gram-negative bacteria which include P. aeruginosa, K. pneumonia, and E. coli³⁶. However, in addition to its antibacterial efficacy β-Myrcene has also been reported to have antioxidant ³⁷, anti-inflammatory³⁸, anti-aging³⁹, anti-nociceptive⁴⁰, activities as well. Therefore, the presence of these highly bioactive compounds in Timur oil provoked us to determine its antibacterial activity.

After the chemical profiling of the Timur seedsoil, we moved on to determine its antibacterial effect against E. coli, S. aureus, P. aeruginosa and B. cereus. The first step was to determine the zone of the inhibition which was carried out by agar well diffusion method. The results of the zone of inhibition are depicted in Table 2. Timur seed essential oil showed 18mm, 15mm, 13mm and 14mm zone of inhibition at 2.5% of concentration against E. coli, S. aureus, P. aeruginosa and B. cereus, respectively. On the other hand, at 5% of concentration Timur seed oil exhibited 22mm, 18mm, 17mm and 18mm zone of inhibition against E. coli, S. aureus, P. aeruginosa and B. cereus, subsequently. The gentamycin used as positive control showed a zone of inhibition of 33mm, 30mm, 27mm and 26mm against E. coli, S. aureus, P. aeruginosa and B. cereus, respectively. Moreover, the minimum inhibitory concentration of Timur seed oil was observed to be 7.5, 15, 7.5 and 5mg/ml against E. coli, S. aureus, P. aeruginosa and B. cereus, correspondingly (Fig 3). The result thus suggests that Timur seeds oil has significant anti-bacterial activity against tested bacteria.

Table 2. Antibacterial activity of Z. armatum seeds essential oil using agar well diffusion method.

S. No	Test Organism	Zone of Inhibition (mm)
S. No	Test Organism	Timur Seeds oil (2.5%)	Timur Seeds oil (5%)	Gentamycin (10mcg/ml)
1.	Escherichia Coli (MTCC 40)	18 ± 1.2	22 ± 1.0	33 ± 1.5
2.	Staphylococcus aureus ((MTCC 7443)	15 ± 1.6	18 ± 1.4	30 ± 2.1
3.	Pseudomonas aeruginosa (MTCC 1668)	13 ± 1.0	17 ± 1.5	27 ± 1.5
4.	Bacillus Cereus (MTCC 6840)	14 ± 1.5	18 ± 2.0	26 ± 1.0

Figure 3. Minimum inhibitory concentration of Z. armatum seed essential oil (mg/ml) against E. coli, S. aureus, P. aeruginosa and B. cereus.

CONCLUSION:

In this study, we demonstrated that the essential oil of Z. armatum (Timur) seeds essential oil has significant antibacterial activity against tested bacteria. We firmly believe that the presence of highly bioactive compounds, i.e., limonene, linalool, trans-Methyl cinnamate and β-Myrceneat a significant concentration in Timur seeds oil accounts for its potent antibacterial activity. However, on the bases of the data obtain we suggest that limonene, linalool and trans-Methyl cinnamateand β-Myrcenerich Timur seedsessential oils can be used in various ways, for example, its efficacy against food borne pathogens (E. coli, P. aeruginosa and B. cereus) suggests that it can be used as preservative in food commodities. Additionally, its toxic effect against S. aureus can be utilized to treat the skin infection and accelerate the wound healing potential.

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Received on 03.11.2023 Modified on 08.04.2024

Research J. Pharm. and Tech 2024; 17(11):5299-5304.

DOI: 10.52711/0974-360X.2024.00811