# Phytochemical Investigation and Antimicrobial Potential of Medicinal PlantNepeta distans Royle ex Benth

Authors: Jawaher Alkahtani; Asma Asma; Muhammad Adil; Abdur Rashid; Turki M. Dawoud; Ahmed A. Alsofi; Mohamed Ragab Abdel Gawwad; Mohamed M. A. Elshaer
Journal: Journal of Food Quality
 (2022)

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## Abstract

Herbal medicines or natural products and plant extract may exhibit promising alternatives or supplements for chemotherapy and antibiotic therapy. The aim of the study is to evaluate the therapeutic value and phytoconstituents of the whole plant,Nepeta distans. The methanol extract contains four known compounds, namely, oleanolic acid, ursolic acid, β-sitosterol, and stigmasterol. The structures of these compounds were confirmed with the help of NMR and mass spectrometry and by comparison with the available literature of these known compounds. The phytochemical analysis test confirmed the presence of alkaloids, flavonoids, saponins, glycosides, fats, proteins, and phytosterols. The antimicrobial activities were carried out by the agar well diffusion method. Both methanol extract and chloroform fraction showed significant antimicrobial activities.


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## Body

## 1. Introduction

Since time unknown, medicinal plants are used for the treatment of different infections. The World Health Organization reports that various plant fractions and their dynamic constituents are utilized as traditional medicines by 80% of the world population [1–4]. Herbal medicines or natural products and plants extract may exhibit promising alternatives or supplements for chemotherapy and antibiotic therapy [5, 6]. Herbal medicines have astonishing potential such as high anticancer, antibacterial, antioxidant, and antifungal activities [7, 8]. Plants offer an inexhaustible source of bioactive compounds and clinically useful drugs for infectious diseases, such as cancer and cardiovascular disorders [9–11]. Bioactive compounds play an important role in drug discovery [12]. However, the increasing antimicrobial resistance to the currently available antimicrobial agents demand intense investigations into the antimicrobial properties of the medicinal plants [13–15]. Regrettably, there has been limited pharmaceutical development of the plants with known bioactivity [16, 17]. There are few pharmaceutical products for infectious diseases that are of plant origin [18, 19]. To fulfill the healthcare needs in Germany and Europe, herbal preparation got more attention, and nearly 1400 herbal preparations are presently used as per European Union [20, 21]. The herbal preparation can also be utilized in various cosmetic industries (as antiwrinkling agents, skin tissue regenerators, and antiage creams) which can lead to an increase in the importance [22, 23].The genusNepeta (Lamiaceae) is a large family which comprises about 400 species, most of which grow in the wild in the central and southern parts of Europe, North Africa, and central and southern Asia. A lot of species of this genus are used in the folk medicine for the antiseptic and astringent properties as topical remedies in children’s cutaneous eruptions and snakes and scorpion bites; orally, they are utilized as antitussive, antispasmodic, antiasthmatic, febrifuge, and diuretic. Moreover, antibacterial, fungicidal, and antiviral activities have been attributed to Nepeta lactones and iridoids contained in several Nepeta species. Some endemic species in the southern Greece are utilized in traditional medicine. In particular, fresh leaves of some Nepeta species are chewed to alleviate toothache and a leaf alcoholic macerate is efficacious for treatment of contusions and rheumatic pains [24]. This genus is also reported to possess biological activities that help in reduction of serum lipids and possess anti-inflammatory, phytotoxic, platelet aggregation, antimicrobial, cytotoxic, and antiglycation properties [25].The aim of the present study is to screen out the bioactive fractions ofNepeta distans, for isolation of the targeted compounds in future.


## 2. Materials and Methods

### 2.1. Plant Collection

The plantNepeta distans was collected from Darra Adam Khel district, Kohat (33.6945°N, 71.4959°E), Pakistan. The collected plants were then identified by a plant taxonomist at the Department of Chemical and Life Sciences, Qurtuba University of Science and Information Technology, Peshawar. It was then cleaned, washed, and dried in the shade. A specimen voucher number (ND-001) was deposited in the herbarium of the same department.


### 2.2. Extraction, Fractionation, and Isolation

The whole plant ofN. distans was grinded to a coarse powder. The powdered plant (2 Kg) was initially extracted with 20 L of 70% MeOH three times at room temperature. It was then filtered and methanol was evaporated under reduced pressure leaving behind a greenish, syrup residue that was dried and weighed. It was 100 g. This MeOH extract was then partitioned into various fractions through a separating funnel. It was partitioned into hexane, chloroform, ethyl acetate, butanol, and water fractions successively. The methanol extract was then subjected to column chromatography which afforded compounds (1–4) from different subfractions at different polarities of the binary solvent system of chloroform/hexane and ethyl acetate/hexane.


### 2.3. Preliminary Phytochemical Qualitative Tests

Alkaloid, flavonoids, saponins, proteins, oils, and glycosides were tested in the crude extract ofNepeta distans using several qualitative tests [26–29].


### 2.4. Antimicrobial Activity

The antifungal and antimicrobial activities of the methanol extract and chloroform fraction ofN. distans were conducted as per available method in the literature [30–34].


#### 2.4.1. Antifungal Activity

Using the agar tube dilution method, the antifungal potential of the methanol extract and chloroform fraction ofNepeta distans was investigated. The stock solution was made with 24 mg extracts per ml (1.0 ml) of sterile DMSO (dimethyl sulfoxide). The Sabouraud dextrose agar medium was made by using a magnetic stirrer to thoroughly dissolve 4.0 g of agar, 4 percent of glucose agar, and 32.5 g of Sabouraud in distilled water (500 ml) to form a homogenous slurry. 4 ml of SDA (Sabouraud dextrose agar) was autoclaved at 120°C for 15 minutes before being chilled to 15°C. When each test tube was injected (4 mm diameter) with fungal strains of 6-7 days old culture for non-mycelia development, an agar surface was created by mixing the stock solution with the nonsolidified SDA medium, and solidifying it in a tilted position, streak was formed. Positive and negative controls were preserved for treatment evaluation by employing DMSO and antifungal medications, respectively (amphotericin B). Subsequent to 7 days of incubation at 27°C, the fungal growth reserve was measured.


#### 2.4.2. Antibacterial Activity

Using the well diffusion agar method, the antibacterial activity of methyl alcohol and chloroform extracts ofNepeta distans was investigated. Bacterial strains were activated by infusing the nutrient broth into the conical flasks and rearing the medium for 24 hours. The agar media was then transferred to Petri dishes containing the injected bacterial strains and solidified under the appropriate conditions. After the medium had solidified, the bores were made in the agar plate using a corkborer, and the extracts (50 l) were transferred to the bore and the plates were reared for 24 hours at 37°C. The results were expressed as the bacterial growth inhibition zone in millimeters. Amoxicillin and ciprofloxacin were employed as positive controls for Gram-positive and Gram-negative bacteria, respectively, and DMSO was utilized as the negative control retain for the purpose of determining the effect.


## 2.1. Plant Collection

The plantNepeta distans was collected from Darra Adam Khel district, Kohat (33.6945°N, 71.4959°E), Pakistan. The collected plants were then identified by a plant taxonomist at the Department of Chemical and Life Sciences, Qurtuba University of Science and Information Technology, Peshawar. It was then cleaned, washed, and dried in the shade. A specimen voucher number (ND-001) was deposited in the herbarium of the same department.


## 2.2. Extraction, Fractionation, and Isolation

The whole plant ofN. distans was grinded to a coarse powder. The powdered plant (2 Kg) was initially extracted with 20 L of 70% MeOH three times at room temperature. It was then filtered and methanol was evaporated under reduced pressure leaving behind a greenish, syrup residue that was dried and weighed. It was 100 g. This MeOH extract was then partitioned into various fractions through a separating funnel. It was partitioned into hexane, chloroform, ethyl acetate, butanol, and water fractions successively. The methanol extract was then subjected to column chromatography which afforded compounds (1–4) from different subfractions at different polarities of the binary solvent system of chloroform/hexane and ethyl acetate/hexane.


## 2.3. Preliminary Phytochemical Qualitative Tests

Alkaloid, flavonoids, saponins, proteins, oils, and glycosides were tested in the crude extract ofNepeta distans using several qualitative tests [26–29].


## 2.4. Antimicrobial Activity

The antifungal and antimicrobial activities of the methanol extract and chloroform fraction ofN. distans were conducted as per available method in the literature [30–34].


### 2.4.1. Antifungal Activity

Using the agar tube dilution method, the antifungal potential of the methanol extract and chloroform fraction ofNepeta distans was investigated. The stock solution was made with 24 mg extracts per ml (1.0 ml) of sterile DMSO (dimethyl sulfoxide). The Sabouraud dextrose agar medium was made by using a magnetic stirrer to thoroughly dissolve 4.0 g of agar, 4 percent of glucose agar, and 32.5 g of Sabouraud in distilled water (500 ml) to form a homogenous slurry. 4 ml of SDA (Sabouraud dextrose agar) was autoclaved at 120°C for 15 minutes before being chilled to 15°C. When each test tube was injected (4 mm diameter) with fungal strains of 6-7 days old culture for non-mycelia development, an agar surface was created by mixing the stock solution with the nonsolidified SDA medium, and solidifying it in a tilted position, streak was formed. Positive and negative controls were preserved for treatment evaluation by employing DMSO and antifungal medications, respectively (amphotericin B). Subsequent to 7 days of incubation at 27°C, the fungal growth reserve was measured.


### 2.4.2. Antibacterial Activity

Using the well diffusion agar method, the antibacterial activity of methyl alcohol and chloroform extracts ofNepeta distans was investigated. Bacterial strains were activated by infusing the nutrient broth into the conical flasks and rearing the medium for 24 hours. The agar media was then transferred to Petri dishes containing the injected bacterial strains and solidified under the appropriate conditions. After the medium had solidified, the bores were made in the agar plate using a corkborer, and the extracts (50 l) were transferred to the bore and the plates were reared for 24 hours at 37°C. The results were expressed as the bacterial growth inhibition zone in millimeters. Amoxicillin and ciprofloxacin were employed as positive controls for Gram-positive and Gram-negative bacteria, respectively, and DMSO was utilized as the negative control retain for the purpose of determining the effect.


## 2.4.1. Antifungal Activity

Using the agar tube dilution method, the antifungal potential of the methanol extract and chloroform fraction ofNepeta distans was investigated. The stock solution was made with 24 mg extracts per ml (1.0 ml) of sterile DMSO (dimethyl sulfoxide). The Sabouraud dextrose agar medium was made by using a magnetic stirrer to thoroughly dissolve 4.0 g of agar, 4 percent of glucose agar, and 32.5 g of Sabouraud in distilled water (500 ml) to form a homogenous slurry. 4 ml of SDA (Sabouraud dextrose agar) was autoclaved at 120°C for 15 minutes before being chilled to 15°C. When each test tube was injected (4 mm diameter) with fungal strains of 6-7 days old culture for non-mycelia development, an agar surface was created by mixing the stock solution with the nonsolidified SDA medium, and solidifying it in a tilted position, streak was formed. Positive and negative controls were preserved for treatment evaluation by employing DMSO and antifungal medications, respectively (amphotericin B). Subsequent to 7 days of incubation at 27°C, the fungal growth reserve was measured.


## 2.4.2. Antibacterial Activity

Using the well diffusion agar method, the antibacterial activity of methyl alcohol and chloroform extracts ofNepeta distans was investigated. Bacterial strains were activated by infusing the nutrient broth into the conical flasks and rearing the medium for 24 hours. The agar media was then transferred to Petri dishes containing the injected bacterial strains and solidified under the appropriate conditions. After the medium had solidified, the bores were made in the agar plate using a corkborer, and the extracts (50 l) were transferred to the bore and the plates were reared for 24 hours at 37°C. The results were expressed as the bacterial growth inhibition zone in millimeters. Amoxicillin and ciprofloxacin were employed as positive controls for Gram-positive and Gram-negative bacteria, respectively, and DMSO was utilized as the negative control retain for the purpose of determining the effect.


## 3. Results and Discussion

### 3.1. Isolation of Phytoconstituents

The methanol extract ofN. distans was subjected to column chromatography and the afforded four compounds were oleanolic acid [35], ursolic acid [36], β-sitosterol [37], and stigmasterol [37]. The structure of these compounds are given in Figure 1.Figure 1
Structures of isolated compounds (1–4).


#### 3.1.1. Spectral Data of Compound(1)

HR-EI-MSm/z: 456.2 [M]+. 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.88 (3H, each s, Me), 3.61 (1H, dd, J = 4.1, 9.91 Hz, H-3), 5.24 (1H, t, J = 3.45 Hz, H-12), 1.12, 1.04, 0.98, 0.97, 0.91. 13C-NMR (CDCl3, 100 MHz) (δ ppm) 15.43 (q, C-25), 15.76 (q, C-24), 17.21 (q, C-26), 18.9 (t, C-6), 23.4 (t, C-16), 23.41 (t, C-11), 23.54 (q, C-30), 25.97 (q, C-27), 27.37 (t, C-2), 27.91 (t, C-15), 28.34 (q, C-23), 30.83 (s, C-20), 33.9 (t, C-21), 42.31 (s, C-14), 32.4 (t, C-22), 183.2 (s, C-28), 32.7 (t, C-7), 33.1 (q, C-29), 37.21 (s, C-10), 143.7 (S, C-13), 38.32 (t, C-1), 38.86 (s, C-4), 39.29 (s, C-8), 121.97 (d, C-12), 41.52 (d, C-18), 45.99 (t, C-19), 48.13 (d, C-9),55.43 (d, C-5), 46.47 (s, C-17), 79.12 (d, C-3).


#### 3.1.2. Spectral Data of Compound(2)

EI-MSm/z (rel. int. %): 456 [M]+ (16), 55 (49), 119 (29), 248 (100), 300 (11), 203 (44), 207 (26), 133 (60), 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.80 (3H, J = 6.8 Hz, Me-29), 0.81 (3H, s, Me-24), 0.86 (3H, s, Me-26), 0.91 (3H, d, J = 6.6 Hz, Me-30), 5.11 (1H, m, H-12), 1.07 (3H, s, Me-23), 0.94 (3H, s, Me-25), 3.19 (1H, dd, Jax, ax = 10.0 Hz, Jax, eq = 4.5 Hz, H-3α), 1.20 (3H, s, Me-27). 13C-NMR (CDCl3, 125 MHz) (δ ppm) 15.4 (q, C-24), 138.7 (s, C-13), 15.9 (q, C-25), (t, C-23, C-30), 125.8 (d, C-12), 22.4 (q, C-29),79.1 (d, C-3), 52.4 (s, C-5), 23.5 (t, C-16), 55.2 (d, C-18), 24.0, 23.9 (t, C-11), 27.4 (t, C-2), 17.2 (q, C-26), 18.3 (t, C-6), 24.5 (q, C-27), 29.4 (t, C-15), 47.9 (s, C-17), 30.5 (d, C-19), 47.4 (d, C-9), 39.6 (s, C-8), 42.0 (s, C-14), 37.0 (t, C-22), 38.5 (t, C-1), 37.1 (s, C-10), 33.2 (t, C-7), 30.3 (d, C-20), 27.5 (t, C-21), 176.2 (s, C-28).


#### 3.1.3. Spectral Data of Compound(3)

EI-MS:m/z: 414 [M]+, 371, 138, 329, 273, 315 and 222. HR-EI-MS: m/z: 414.3857 (C29H50O, 414.3861). 1H-NMR: (CDCl3, 400 MHz) (δ ppm) 0.66 (3H, s, H-18), 0.78 (3H, d, J = 6.0 Hz, H-27), 0.81 (3H, d, J = 6.2 Hz, H-26), 0.98 (3H, s, H-19), 3.35 (1H, m, H-3), 0.82 (3H, t, J = 7.5 Hz, H-29), 0.90 (3H, d, J = 6.5 Hz, H-21), 5.32 (1H, br. s, H-6). 13C-NMR: (CDCl3, 125 MHz) (δ ppm) 11.8 (C-18), 121.3 (C-6), 142.0 (C-5), 26.2 (C-16), 70.3 (C-3), 56.9 (C-14), 56.7 (C-17), 34.0 (C-22), 50.3 (C-9), 32.1 (C-8), 49.0 (C-24), 42.3 (C-13), 39.9 (C-12), 40.2 (C-4), 37.3 (C-1), 37.1 (C-10), 31.6 (C-7), 28.9 (C-23, C-25), 28.6 (C-2), 22.8 (C-28), 25.3 (C-15), 20.1 (C-26), 21.1 (C-11), 19.1 (C-19), 19.3 (C-27), 18.6 (C-210), 12.0 (C-29).


#### 3.1.4. Spectral Data of Compound(4)

HR-EI-MSm/z: 412.3920. Calcd. for C29H48O, 412.3926 EI-MS (rel. int. %) m/z: M+ 412 (7), 270 (22), 379 (28), 394 (20), 397 (12), 273 (30), 327 (60), 300 (67), 369 (35), 351 (70), 301 (18). 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.65 (3H, s, Me-18), 0.80 (3H, s, Me-19), 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.84 (3H, t, J = 7.0 Hz, Me-29), 0.90 (3H, d, J = 6.5 Hz, Me-21), 5.33 (1H, m, H-6), 5.02 (1H, m, H-3), 5.15 (1H, dd, J = 15.1, 8.4 Hz, H-22), 0.83 (1H, d, J = 6.6 Hz, Me-26), 13C-NMR (CDCl3, 100 MHz) (δ ppm) 12.0 (C-29), 129.4 (C-23), 19.4 (C-19), 21.0 (C-11), 31.8(C-2), 32.0 (C-25), 42.4(C-13), C-24), 39.7, 25.4 (C-28), 24.4 (C-15), 121.7 (C-6), 51.3 ((C-12), 71.9 (C-3), 28.9 (C-16), 40.5 (C-20), 50.3 (C-9), 42.2 (C-4), 32.2 (C-8), 21.2 (C-27), 57.0 (C-14), 56.0 (C-17), 36.6 (C-10), 37.4 (C-1), 31.9 (C-7), 21.1 (C-21), 19.0 (C-26), 138.4 (C-22), 12.4 (C-18), and 140.9 (C-5).


### 3.2. Antimicrobial Activities

#### 3.2.1. Antibacterial Potential

The result of the antibacterial activity shows that methanol extract ofNepeta distans shows the maximum region of inhibition (33 mm) against Klebsiella pneumoniae at 300 mg/ml, while the lowest inhibition of 22.5 mm was shown against Escherichia coli at 100 mg/ml (Table 1). Similarly, the chloroform extract of Nepeta distans showed the highest zone of inhibition (30 mm) against Staphylococcus aureus 300 mg/ml, while the lowest zone of inhibition (21 mm) was shown against E. coli at 100 mg/ml (Table 1). The methanol extract showed the lowest MIC values (2 μg/ml) against E. coli and pathovar, and the chloroform extract showed the lowest MIC values (2 μg/ml) against S. aureus (Table 2).The MIC values are shown in Table 2.Table 1
Antibacterial activity of the methanol extract and chloroform fraction ofN. distans.
Test organismPositive controlNegative controlMethanol extract (mg/ml)Chloroform fraction (mg/ml)Concentration100200300100200300Zone of inhibition (mm)E. coli10022.526.527212528Pathovar100243029242927S. aureus10024.52932222730K. pneumoniae10025.52933232928Table 2
MIC values (gm/ml) of the antibacterial activity of methanol extract and chloroform fraction ofN. distans.
Test organismMethanol extract (mg/ml)Chloroform fraction (mg/ml)E. coli24S. aureus62K. pneumoniae46Pathovar24


#### 3.2.2. Antifungal Activity

The result of the antifungal activity show that methanol extract ofNepeta distans show the highest zone of inhibition (35 mm) at 300 mg/ml, while the lowest zone of inhibition (22.5 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). Similarly, the chloroform extract of the Nepeta distans showed the highest zone of inhibition (31 mm) against Penicillium chrysogenum at 300 mg/ml, while the lowest zone of inhibition (23 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). The methanol extract showed the lowest MIC values (2 μg/ml) against Aspergillus niger, and the chloroform extract showed the lowest MIC values (2 μg/ml) against Aspergillus tamarii. Both the methanol extract and the chloroform fraction showed moderate activities against Aspergillus flavus and Aspergillus fumigatus at all three concentrations (Table 4). The MIC values are displayed in Table 4.Table 3
Antifungal activity of the methanol extract and chloroform fraction ofN. distans.
Test organismPositive controlNegative controlMethanol extract (mg/ml)Chloroform extract (mg/ml)Concentration100200300100200300Zone of inhibition (mm)A. flavus10027.52926282521A. fumigatus10025.524.523272924F. solani1002724.522.5302722A. Niger1003328.524.5282923P. chrysogenum100353228.5313028A. tamarii100322925302722Table 4
MIC values (gm/ml) of the antifungal activity of the methanol extract and chloroform fraction ofN. distans.
Test organismMethanol extract (mg/ml)Chloroform extract (mg/ml)A. flavus24A. fumigatus62F. solani46A. Niger24P. chrysogenum42A. tamarii64


### 3.3. Phytochemical Screening

Phytochemical screening showed that both methanol extract and chloroform fraction showed the presence of alkaloids, flavonoids, saponins, glycosides, proteins, and oil (Table5).Table 5
Qualitative phytochemical analysis ofNepeta distans.
Class of phytochemicalMethanol extractChloroform fractionAlkaloids++Flavonoids++Saponins++Proteins++Oils++Glycosides++Screening the crude extract for phytochemical analysis is an imperative means for finding the secondary metabolites that exist in the medicinal plant because these phytochemicals are counted responsible for therapeutic application. These phytochemicals responsible for that it has been reported that saponins are among the most important compounds responsible for antidiabetic, antispasmodic, antitumor, anthelmintic, antimicrobial, phytotoxic, cytotoxic, and antioxidant potential [38].Nepeta distans is a rich source for natural products; many constituents isolated and reported in literature are β-sitosterol, eugenol, ursolic acid, thymoquinone, oleanolic acid, nepedinol, netidiol, markhamioside F, and nepatanol [39].A study confirmed that the genusNepeta species is an important source of nutrients and showed immunomodulatory potential. Nepeta is multiregional genus and a variety of compounds are reported from it in literature, such as β-amyrin, glutinol, stigmasterol glucoside, 9β, 10α-cleroda-3, 13 (16),14-trien-18-oic, 5, 4′-dihydroxy-3, 6, 7-trimethoxyflavone, (−)-6β-hydroxy-15, 16-epoxy-5β, 8β ,and stigmasterol [40–42]. The isolation and characterization of the compounds 1–4 from Nepeta distans are in complete agreement with the findings of [40–42]. Similarly, many species of the genus Nepeta are used traditionally in folklore medicine for the treatment of liver and kidney problems, to treat dysentery and teeth problems, and are used as sedative agents and stimulants, and they are also used as febrifuge, antiasthmatic, antispasmodic, diaphoretic, and diuretic.Essential oil and crude extracts ofNepeta genus have showed antimicrobial potential, significant antiglycation activity, anti-inflammatory potential, and serum lipids reduction potential [43]. Our findings of antimicrobial potential of Nepeta dastans are again completely in agreement with the published literature [43]. Another published research showed that the Nepeta species possessed multibiological potentials such as antidiabetic, acetylcholinesterase inhibitory, antiatherosclerotic, analgesic, anti-ociceptive, antimalarial, antileishmanial, antioxidant, antimicrobial, anthelmintic, antiglycation, antiplatelet aggregation, cardioprotective effect, cytotoxic, anticancer and apoptotic, genotoxic, immunomodulatory, hepatoprotective, insecticidal and insect repellent, trypanocidal, phytotoxic, nematocidal, dyslipidemia, and larvicidal [44].


## 3.1. Isolation of Phytoconstituents

The methanol extract ofN. distans was subjected to column chromatography and the afforded four compounds were oleanolic acid [35], ursolic acid [36], β-sitosterol [37], and stigmasterol [37]. The structure of these compounds are given in Figure 1.Figure 1
Structures of isolated compounds (1–4).


### 3.1.1. Spectral Data of Compound(1)

HR-EI-MSm/z: 456.2 [M]+. 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.88 (3H, each s, Me), 3.61 (1H, dd, J = 4.1, 9.91 Hz, H-3), 5.24 (1H, t, J = 3.45 Hz, H-12), 1.12, 1.04, 0.98, 0.97, 0.91. 13C-NMR (CDCl3, 100 MHz) (δ ppm) 15.43 (q, C-25), 15.76 (q, C-24), 17.21 (q, C-26), 18.9 (t, C-6), 23.4 (t, C-16), 23.41 (t, C-11), 23.54 (q, C-30), 25.97 (q, C-27), 27.37 (t, C-2), 27.91 (t, C-15), 28.34 (q, C-23), 30.83 (s, C-20), 33.9 (t, C-21), 42.31 (s, C-14), 32.4 (t, C-22), 183.2 (s, C-28), 32.7 (t, C-7), 33.1 (q, C-29), 37.21 (s, C-10), 143.7 (S, C-13), 38.32 (t, C-1), 38.86 (s, C-4), 39.29 (s, C-8), 121.97 (d, C-12), 41.52 (d, C-18), 45.99 (t, C-19), 48.13 (d, C-9),55.43 (d, C-5), 46.47 (s, C-17), 79.12 (d, C-3).


### 3.1.2. Spectral Data of Compound(2)

EI-MSm/z (rel. int. %): 456 [M]+ (16), 55 (49), 119 (29), 248 (100), 300 (11), 203 (44), 207 (26), 133 (60), 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.80 (3H, J = 6.8 Hz, Me-29), 0.81 (3H, s, Me-24), 0.86 (3H, s, Me-26), 0.91 (3H, d, J = 6.6 Hz, Me-30), 5.11 (1H, m, H-12), 1.07 (3H, s, Me-23), 0.94 (3H, s, Me-25), 3.19 (1H, dd, Jax, ax = 10.0 Hz, Jax, eq = 4.5 Hz, H-3α), 1.20 (3H, s, Me-27). 13C-NMR (CDCl3, 125 MHz) (δ ppm) 15.4 (q, C-24), 138.7 (s, C-13), 15.9 (q, C-25), (t, C-23, C-30), 125.8 (d, C-12), 22.4 (q, C-29),79.1 (d, C-3), 52.4 (s, C-5), 23.5 (t, C-16), 55.2 (d, C-18), 24.0, 23.9 (t, C-11), 27.4 (t, C-2), 17.2 (q, C-26), 18.3 (t, C-6), 24.5 (q, C-27), 29.4 (t, C-15), 47.9 (s, C-17), 30.5 (d, C-19), 47.4 (d, C-9), 39.6 (s, C-8), 42.0 (s, C-14), 37.0 (t, C-22), 38.5 (t, C-1), 37.1 (s, C-10), 33.2 (t, C-7), 30.3 (d, C-20), 27.5 (t, C-21), 176.2 (s, C-28).


### 3.1.3. Spectral Data of Compound(3)

EI-MS:m/z: 414 [M]+, 371, 138, 329, 273, 315 and 222. HR-EI-MS: m/z: 414.3857 (C29H50O, 414.3861). 1H-NMR: (CDCl3, 400 MHz) (δ ppm) 0.66 (3H, s, H-18), 0.78 (3H, d, J = 6.0 Hz, H-27), 0.81 (3H, d, J = 6.2 Hz, H-26), 0.98 (3H, s, H-19), 3.35 (1H, m, H-3), 0.82 (3H, t, J = 7.5 Hz, H-29), 0.90 (3H, d, J = 6.5 Hz, H-21), 5.32 (1H, br. s, H-6). 13C-NMR: (CDCl3, 125 MHz) (δ ppm) 11.8 (C-18), 121.3 (C-6), 142.0 (C-5), 26.2 (C-16), 70.3 (C-3), 56.9 (C-14), 56.7 (C-17), 34.0 (C-22), 50.3 (C-9), 32.1 (C-8), 49.0 (C-24), 42.3 (C-13), 39.9 (C-12), 40.2 (C-4), 37.3 (C-1), 37.1 (C-10), 31.6 (C-7), 28.9 (C-23, C-25), 28.6 (C-2), 22.8 (C-28), 25.3 (C-15), 20.1 (C-26), 21.1 (C-11), 19.1 (C-19), 19.3 (C-27), 18.6 (C-210), 12.0 (C-29).


### 3.1.4. Spectral Data of Compound(4)

HR-EI-MSm/z: 412.3920. Calcd. for C29H48O, 412.3926 EI-MS (rel. int. %) m/z: M+ 412 (7), 270 (22), 379 (28), 394 (20), 397 (12), 273 (30), 327 (60), 300 (67), 369 (35), 351 (70), 301 (18). 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.65 (3H, s, Me-18), 0.80 (3H, s, Me-19), 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.84 (3H, t, J = 7.0 Hz, Me-29), 0.90 (3H, d, J = 6.5 Hz, Me-21), 5.33 (1H, m, H-6), 5.02 (1H, m, H-3), 5.15 (1H, dd, J = 15.1, 8.4 Hz, H-22), 0.83 (1H, d, J = 6.6 Hz, Me-26), 13C-NMR (CDCl3, 100 MHz) (δ ppm) 12.0 (C-29), 129.4 (C-23), 19.4 (C-19), 21.0 (C-11), 31.8(C-2), 32.0 (C-25), 42.4(C-13), C-24), 39.7, 25.4 (C-28), 24.4 (C-15), 121.7 (C-6), 51.3 ((C-12), 71.9 (C-3), 28.9 (C-16), 40.5 (C-20), 50.3 (C-9), 42.2 (C-4), 32.2 (C-8), 21.2 (C-27), 57.0 (C-14), 56.0 (C-17), 36.6 (C-10), 37.4 (C-1), 31.9 (C-7), 21.1 (C-21), 19.0 (C-26), 138.4 (C-22), 12.4 (C-18), and 140.9 (C-5).


## 3.1.1. Spectral Data of Compound(1)

HR-EI-MSm/z: 456.2 [M]+. 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.88 (3H, each s, Me), 3.61 (1H, dd, J = 4.1, 9.91 Hz, H-3), 5.24 (1H, t, J = 3.45 Hz, H-12), 1.12, 1.04, 0.98, 0.97, 0.91. 13C-NMR (CDCl3, 100 MHz) (δ ppm) 15.43 (q, C-25), 15.76 (q, C-24), 17.21 (q, C-26), 18.9 (t, C-6), 23.4 (t, C-16), 23.41 (t, C-11), 23.54 (q, C-30), 25.97 (q, C-27), 27.37 (t, C-2), 27.91 (t, C-15), 28.34 (q, C-23), 30.83 (s, C-20), 33.9 (t, C-21), 42.31 (s, C-14), 32.4 (t, C-22), 183.2 (s, C-28), 32.7 (t, C-7), 33.1 (q, C-29), 37.21 (s, C-10), 143.7 (S, C-13), 38.32 (t, C-1), 38.86 (s, C-4), 39.29 (s, C-8), 121.97 (d, C-12), 41.52 (d, C-18), 45.99 (t, C-19), 48.13 (d, C-9),55.43 (d, C-5), 46.47 (s, C-17), 79.12 (d, C-3).


## 3.1.2. Spectral Data of Compound(2)

EI-MSm/z (rel. int. %): 456 [M]+ (16), 55 (49), 119 (29), 248 (100), 300 (11), 203 (44), 207 (26), 133 (60), 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.80 (3H, J = 6.8 Hz, Me-29), 0.81 (3H, s, Me-24), 0.86 (3H, s, Me-26), 0.91 (3H, d, J = 6.6 Hz, Me-30), 5.11 (1H, m, H-12), 1.07 (3H, s, Me-23), 0.94 (3H, s, Me-25), 3.19 (1H, dd, Jax, ax = 10.0 Hz, Jax, eq = 4.5 Hz, H-3α), 1.20 (3H, s, Me-27). 13C-NMR (CDCl3, 125 MHz) (δ ppm) 15.4 (q, C-24), 138.7 (s, C-13), 15.9 (q, C-25), (t, C-23, C-30), 125.8 (d, C-12), 22.4 (q, C-29),79.1 (d, C-3), 52.4 (s, C-5), 23.5 (t, C-16), 55.2 (d, C-18), 24.0, 23.9 (t, C-11), 27.4 (t, C-2), 17.2 (q, C-26), 18.3 (t, C-6), 24.5 (q, C-27), 29.4 (t, C-15), 47.9 (s, C-17), 30.5 (d, C-19), 47.4 (d, C-9), 39.6 (s, C-8), 42.0 (s, C-14), 37.0 (t, C-22), 38.5 (t, C-1), 37.1 (s, C-10), 33.2 (t, C-7), 30.3 (d, C-20), 27.5 (t, C-21), 176.2 (s, C-28).


## 3.1.3. Spectral Data of Compound(3)

EI-MS:m/z: 414 [M]+, 371, 138, 329, 273, 315 and 222. HR-EI-MS: m/z: 414.3857 (C29H50O, 414.3861). 1H-NMR: (CDCl3, 400 MHz) (δ ppm) 0.66 (3H, s, H-18), 0.78 (3H, d, J = 6.0 Hz, H-27), 0.81 (3H, d, J = 6.2 Hz, H-26), 0.98 (3H, s, H-19), 3.35 (1H, m, H-3), 0.82 (3H, t, J = 7.5 Hz, H-29), 0.90 (3H, d, J = 6.5 Hz, H-21), 5.32 (1H, br. s, H-6). 13C-NMR: (CDCl3, 125 MHz) (δ ppm) 11.8 (C-18), 121.3 (C-6), 142.0 (C-5), 26.2 (C-16), 70.3 (C-3), 56.9 (C-14), 56.7 (C-17), 34.0 (C-22), 50.3 (C-9), 32.1 (C-8), 49.0 (C-24), 42.3 (C-13), 39.9 (C-12), 40.2 (C-4), 37.3 (C-1), 37.1 (C-10), 31.6 (C-7), 28.9 (C-23, C-25), 28.6 (C-2), 22.8 (C-28), 25.3 (C-15), 20.1 (C-26), 21.1 (C-11), 19.1 (C-19), 19.3 (C-27), 18.6 (C-210), 12.0 (C-29).


## 3.1.4. Spectral Data of Compound(4)

HR-EI-MSm/z: 412.3920. Calcd. for C29H48O, 412.3926 EI-MS (rel. int. %) m/z: M+ 412 (7), 270 (22), 379 (28), 394 (20), 397 (12), 273 (30), 327 (60), 300 (67), 369 (35), 351 (70), 301 (18). 1H-NMR (CDCl3, 400 MHz) (δ ppm) 0.65 (3H, s, Me-18), 0.80 (3H, s, Me-19), 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.84 (3H, t, J = 7.0 Hz, Me-29), 0.90 (3H, d, J = 6.5 Hz, Me-21), 5.33 (1H, m, H-6), 5.02 (1H, m, H-3), 5.15 (1H, dd, J = 15.1, 8.4 Hz, H-22), 0.83 (1H, d, J = 6.6 Hz, Me-26), 13C-NMR (CDCl3, 100 MHz) (δ ppm) 12.0 (C-29), 129.4 (C-23), 19.4 (C-19), 21.0 (C-11), 31.8(C-2), 32.0 (C-25), 42.4(C-13), C-24), 39.7, 25.4 (C-28), 24.4 (C-15), 121.7 (C-6), 51.3 ((C-12), 71.9 (C-3), 28.9 (C-16), 40.5 (C-20), 50.3 (C-9), 42.2 (C-4), 32.2 (C-8), 21.2 (C-27), 57.0 (C-14), 56.0 (C-17), 36.6 (C-10), 37.4 (C-1), 31.9 (C-7), 21.1 (C-21), 19.0 (C-26), 138.4 (C-22), 12.4 (C-18), and 140.9 (C-5).


## 3.2. Antimicrobial Activities

### 3.2.1. Antibacterial Potential

The result of the antibacterial activity shows that methanol extract ofNepeta distans shows the maximum region of inhibition (33 mm) against Klebsiella pneumoniae at 300 mg/ml, while the lowest inhibition of 22.5 mm was shown against Escherichia coli at 100 mg/ml (Table 1). Similarly, the chloroform extract of Nepeta distans showed the highest zone of inhibition (30 mm) against Staphylococcus aureus 300 mg/ml, while the lowest zone of inhibition (21 mm) was shown against E. coli at 100 mg/ml (Table 1). The methanol extract showed the lowest MIC values (2 μg/ml) against E. coli and pathovar, and the chloroform extract showed the lowest MIC values (2 μg/ml) against S. aureus (Table 2).The MIC values are shown in Table 2.Table 1
Antibacterial activity of the methanol extract and chloroform fraction ofN. distans.
Test organismPositive controlNegative controlMethanol extract (mg/ml)Chloroform fraction (mg/ml)Concentration100200300100200300Zone of inhibition (mm)E. coli10022.526.527212528Pathovar100243029242927S. aureus10024.52932222730K. pneumoniae10025.52933232928Table 2
MIC values (gm/ml) of the antibacterial activity of methanol extract and chloroform fraction ofN. distans.
Test organismMethanol extract (mg/ml)Chloroform fraction (mg/ml)E. coli24S. aureus62K. pneumoniae46Pathovar24


### 3.2.2. Antifungal Activity

The result of the antifungal activity show that methanol extract ofNepeta distans show the highest zone of inhibition (35 mm) at 300 mg/ml, while the lowest zone of inhibition (22.5 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). Similarly, the chloroform extract of the Nepeta distans showed the highest zone of inhibition (31 mm) against Penicillium chrysogenum at 300 mg/ml, while the lowest zone of inhibition (23 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). The methanol extract showed the lowest MIC values (2 μg/ml) against Aspergillus niger, and the chloroform extract showed the lowest MIC values (2 μg/ml) against Aspergillus tamarii. Both the methanol extract and the chloroform fraction showed moderate activities against Aspergillus flavus and Aspergillus fumigatus at all three concentrations (Table 4). The MIC values are displayed in Table 4.Table 3
Antifungal activity of the methanol extract and chloroform fraction ofN. distans.
Test organismPositive controlNegative controlMethanol extract (mg/ml)Chloroform extract (mg/ml)Concentration100200300100200300Zone of inhibition (mm)A. flavus10027.52926282521A. fumigatus10025.524.523272924F. solani1002724.522.5302722A. Niger1003328.524.5282923P. chrysogenum100353228.5313028A. tamarii100322925302722Table 4
MIC values (gm/ml) of the antifungal activity of the methanol extract and chloroform fraction ofN. distans.
Test organismMethanol extract (mg/ml)Chloroform extract (mg/ml)A. flavus24A. fumigatus62F. solani46A. Niger24P. chrysogenum42A. tamarii64


## 3.2.1. Antibacterial Potential

The result of the antibacterial activity shows that methanol extract ofNepeta distans shows the maximum region of inhibition (33 mm) against Klebsiella pneumoniae at 300 mg/ml, while the lowest inhibition of 22.5 mm was shown against Escherichia coli at 100 mg/ml (Table 1). Similarly, the chloroform extract of Nepeta distans showed the highest zone of inhibition (30 mm) against Staphylococcus aureus 300 mg/ml, while the lowest zone of inhibition (21 mm) was shown against E. coli at 100 mg/ml (Table 1). The methanol extract showed the lowest MIC values (2 μg/ml) against E. coli and pathovar, and the chloroform extract showed the lowest MIC values (2 μg/ml) against S. aureus (Table 2).The MIC values are shown in Table 2.Table 1
Antibacterial activity of the methanol extract and chloroform fraction ofN. distans.
Test organismPositive controlNegative controlMethanol extract (mg/ml)Chloroform fraction (mg/ml)Concentration100200300100200300Zone of inhibition (mm)E. coli10022.526.527212528Pathovar100243029242927S. aureus10024.52932222730K. pneumoniae10025.52933232928Table 2
MIC values (gm/ml) of the antibacterial activity of methanol extract and chloroform fraction ofN. distans.
Test organismMethanol extract (mg/ml)Chloroform fraction (mg/ml)E. coli24S. aureus62K. pneumoniae46Pathovar24


## 3.2.2. Antifungal Activity

The result of the antifungal activity show that methanol extract ofNepeta distans show the highest zone of inhibition (35 mm) at 300 mg/ml, while the lowest zone of inhibition (22.5 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). Similarly, the chloroform extract of the Nepeta distans showed the highest zone of inhibition (31 mm) against Penicillium chrysogenum at 300 mg/ml, while the lowest zone of inhibition (23 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). The methanol extract showed the lowest MIC values (2 μg/ml) against Aspergillus niger, and the chloroform extract showed the lowest MIC values (2 μg/ml) against Aspergillus tamarii. Both the methanol extract and the chloroform fraction showed moderate activities against Aspergillus flavus and Aspergillus fumigatus at all three concentrations (Table 4). The MIC values are displayed in Table 4.Table 3
Antifungal activity of the methanol extract and chloroform fraction ofN. distans.
Test organismPositive controlNegative controlMethanol extract (mg/ml)Chloroform extract (mg/ml)Concentration100200300100200300Zone of inhibition (mm)A. flavus10027.52926282521A. fumigatus10025.524.523272924F. solani1002724.522.5302722A. Niger1003328.524.5282923P. chrysogenum100353228.5313028A. tamarii100322925302722Table 4
MIC values (gm/ml) of the antifungal activity of the methanol extract and chloroform fraction ofN. distans.
Test organismMethanol extract (mg/ml)Chloroform extract (mg/ml)A. flavus24A. fumigatus62F. solani46A. Niger24P. chrysogenum42A. tamarii64


## 3.3. Phytochemical Screening

Phytochemical screening showed that both methanol extract and chloroform fraction showed the presence of alkaloids, flavonoids, saponins, glycosides, proteins, and oil (Table5).Table 5
Qualitative phytochemical analysis ofNepeta distans.
Class of phytochemicalMethanol extractChloroform fractionAlkaloids++Flavonoids++Saponins++Proteins++Oils++Glycosides++Screening the crude extract for phytochemical analysis is an imperative means for finding the secondary metabolites that exist in the medicinal plant because these phytochemicals are counted responsible for therapeutic application. These phytochemicals responsible for that it has been reported that saponins are among the most important compounds responsible for antidiabetic, antispasmodic, antitumor, anthelmintic, antimicrobial, phytotoxic, cytotoxic, and antioxidant potential [38].Nepeta distans is a rich source for natural products; many constituents isolated and reported in literature are β-sitosterol, eugenol, ursolic acid, thymoquinone, oleanolic acid, nepedinol, netidiol, markhamioside F, and nepatanol [39].A study confirmed that the genusNepeta species is an important source of nutrients and showed immunomodulatory potential. Nepeta is multiregional genus and a variety of compounds are reported from it in literature, such as β-amyrin, glutinol, stigmasterol glucoside, 9β, 10α-cleroda-3, 13 (16),14-trien-18-oic, 5, 4′-dihydroxy-3, 6, 7-trimethoxyflavone, (−)-6β-hydroxy-15, 16-epoxy-5β, 8β ,and stigmasterol [40–42]. The isolation and characterization of the compounds 1–4 from Nepeta distans are in complete agreement with the findings of [40–42]. Similarly, many species of the genus Nepeta are used traditionally in folklore medicine for the treatment of liver and kidney problems, to treat dysentery and teeth problems, and are used as sedative agents and stimulants, and they are also used as febrifuge, antiasthmatic, antispasmodic, diaphoretic, and diuretic.Essential oil and crude extracts ofNepeta genus have showed antimicrobial potential, significant antiglycation activity, anti-inflammatory potential, and serum lipids reduction potential [43]. Our findings of antimicrobial potential of Nepeta dastans are again completely in agreement with the published literature [43]. Another published research showed that the Nepeta species possessed multibiological potentials such as antidiabetic, acetylcholinesterase inhibitory, antiatherosclerotic, analgesic, anti-ociceptive, antimalarial, antileishmanial, antioxidant, antimicrobial, anthelmintic, antiglycation, antiplatelet aggregation, cardioprotective effect, cytotoxic, anticancer and apoptotic, genotoxic, immunomodulatory, hepatoprotective, insecticidal and insect repellent, trypanocidal, phytotoxic, nematocidal, dyslipidemia, and larvicidal [44].


## 4. Conclusion

The present study shows thatNepeta distans possess bioactive phytochemicals such as flavonoids, alkaloids, and tannins , and its methanol extract and chloroform fraction showed antimicrobial potential. The published literature showed multiple biological activities of Nepeta species. Therefore, it is highly recommended to explore Nepeta distans for more phytochemical as well as biological investigation.



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*Source: 8386326-2022-08-17.xml*