Phytochemical and Antimicrobial Potential of the Roots of Berberis brevissima Jafri. (Berberidaceae)

Berberis is the only genus of the family Berberidaceae, in the southerly hemisphere and comprises 600 species (Bai et al., 2011; Ali et al., 2020). About 24 species are widely distributed in Pakistan’s mountainous parts, including Baluchistan, Gilgit, Chitral, Hazara, Murree, Swat, Dir, and Kashmir (Ali et al., 2020). The species is about 1-5 m tall, having deciduous and evergreen shrubs with yellow wood, thorny shoots, and simple leaves. Many species are distinguished for their attractive pink, violet, blue, or dark red color (Arayne et al., 2007). Various species of Berberis are generally dispersed in the temperate and semi-temperate region and are inherent of the whole range of the Himalayas Mountains and North America, Europe, Asia, and the Mediterranean regions (Knapp and Melly, 1986; Chevallier, 2011). The Species of the Berberis genus are famous for their traditional medicinal use. The evergreen shrub species with the intense pale-yellow color of shoots and roots are used for various ailments, such as rheumatism, stomach disorders diabetes, ear and eye infections, malarial fever, fever, skin disease, and jaundice in remote and rural areas (Ambastha, 1988; Watt, 1989; Whittemore, 1997; Janbaz and Gilani et al., 2000; Srivastava et al., 2006; Boon and Smith, 2009; Hassan et al., 2010; Godfrey et al., 2011). The extracts obtained from Berberis leaves were revealed to cure dysentery, scurvy, sore throat, angina, and have been used in modern drug preparations (Mokhber-Dezfuli et al., 2014; Ali et al., 2020).

Lately isolates from berberis species (berberine, sparteine, and scopolamine) have been employed for the allergic inflammation of the eye, the metabolic capacity of man and for the prevention of travel sickness, respectively (Souto et al., 2011). Berberine is an alkaloid present in roots, young shoots, leaves, flowers, fruits, and stems of all Berberis species and is available as medicine. The berberine content increases as the plant ages (Ali et al., 2015). Furthermore, berberine is a therapeutic alkaloid and has antidiabetic (Steriti, 2010), antidiarrheal (Issat et al., 2006), antitumor (Issat et al., 2006), anti-hyper cholesterol mia (Doggrell, 2005), anti-inflammatory (Kuo et al., 2004), and antipyretic (Küpeli et al., 2002) properties.

Assyrians used the fruits of Berberis as blood purifying agents (Karimov, 1993). The anticancer potential of B. aristata has been reported (Das et al., 2009). Various types of ulcers have been cured by using extracts obtained from the roots of B. asiatica, B. aristate, and B. lycium (Singh, 2007), also these extracts revealed antifungal activities.

B. brevissima locally is known as “ziar largy” and Barberry, found at Tirah (Khyber agency) in the northwest side of Pakistan. Roots, aerial parts, and especially the fruits are used for various medicinal and dietary purposes. The raw powdered and its crude extracts are used as tonic, astringent, and diaphoretic agents. The dietary uses include the fruit portion in the preparation of sauces, jellies, wines, and many other types of juices. It is also used as preservatives in many foods, and its pale-yellow color of roots and bark is an indigenous source of dye for female clothes. Recently many different products have been developed from B. brevissima, on a commercial scale (Kupeli et al., 2002). In Iran, this plant is used commonly in rice pilafs and as a flavoring agent for poultry meat. Their edible purple fruits are used for jams and infusions (Heywood and Chant, 1982). Research indicated that this plant is particularly active against cholera, giardia, shigella, salmonella, and E. coli (Chevallier, 2011).

Due to the advancement in spectroscopy (Nuclear magnetic resonance spectroscopy, mass spectroscopy, and X-Ray crystallography), elucidation and characterization of natural products have become much easier. Keeping the importance of native medicinal plants, the current study was conducted on the ethyl acetate fraction (Fr. C) of Berberis brevissima Jafri to investigate possible antibacterial and fatty acids profile of the n-hexane oil fractions using GC-MS, and purification of bioactive compounds.

The Berberis brevissima Jafri was collected on 25th July 2010 from Tirrah (Khyber Agency). Identification of the plant was carried out with specimen # Bot/10710 by plant taxonomist Prof. Dr. Jandar Shah, Benazir Bhutto University Sherengal Campus Upper Dir.

The dried and ground roots of Berberis brevissima Jafri (5 Kg) were soaked in methanol for one week (10 L x 7 days). The crude extract (118.8 g) was collected in flasks and recovered using a rotary evaporator at 45 °C temperature.

The obtained MeOH crude extract (118.8 g) from the roots of B. brevissima was further fractionated for obtaining alkaloidal fraction through acidification and basification process. The water was acidified (pH=2) with the addition of HCl. pH paper was used to adjust the pH. In 1L of this acidified water, the crude MeOH extract was dissolved. The undissolved residue was collected and named Fr. A (23.5 g). The remaining H2O soluble uniform mixture was shifted to a separating flask, and dichloromethane (3×500 mL) was added, shaken well, and allowed for the split of two layers. The dichloromethane layer was collected as Fr. B (21.8 g). Afterward the remaining homogenous water residue was basified with the addition of aqueous ammonia (pH = 8) and obtained fraction Fr. C (27.4 g) with the addition of ethyl acetate (3×500 mL). The remaining water residue was collected as Fr. D (46.1 g).

The ethyl acetate fraction (Fr. C) was further processed through open gravity silica gel column chromatography (CC), and elution was carried out with n-hexane, chloroform, and methanol in a gradient manner. 57 sub-fractions were obtained from column chromatography (CC) based on TLC profile similarity. Sub-fraction Z-1 was further fractionated using CC and got six oil sub-fractions (H-1 – H-6). Sub-fraction Z-4 was further purified on a small Si-gel column, and compound 1 was isolated. Sub-fractions Z-42 and Z-46 were further purified through small columns and yielded compounds 2 and 3, respectively.

The six oil sub-fractions were analyzed by GC-MS. 1.5 mL of 0.5 M methanolic NaOH was added to the flask containing 25.0 mg of the sample in a 25 mL volumetric flask and heated at 50 °C for five minutes in a water bath. After that, 1.5 mL of BF3-MeOH was added and the mixture was heated at 80 °C for 5 min. Then, a saturated NaCl solution (2 mL) was added to the flask. Afterward, the sample mixture was moved to a separatory funnel, and added 5 mL of n-hexane, shaken, and allowed for the separation of the aqueous and organic layers. The n-hexane layer (esterified layer) was removed and transferred into a 50 mL flask through filter paper. (Tokul-Olmez et al., 2018).

For GC-MS analysis (QP 2010 plus-Shimadzu, Tokyo-Japan) of fatty acids, a capillary column (30 m, 0.35 mm, 0.250 µm) with 100Pka pressure was used. The initial temperature of the column was kept at 50 °C for one minute, then at the rate of 15 °C/min, raised to 150 °C, then at the rate of 2.5 °C/min, raised to 175 °C for 5 min, and finally raised to 220 °C for 5 min, the total run time was 45 min. Interface and ion source temperatures were 240 °C and 250 °C, respectively. The sample (1 μL) was inserted at 240 °C. Helium was used as a sample carrier.

EI-MS were taken at ionization energy of 70 eV, with the mass scanning range from m/z 85 to m/z 380 amu. NIST (NIST 05) library was used for compounds identification.

The antibacterial potential of the oil sub-fractions (H-1 to H-6) was carried out through the Agar Well Diffusion assay. At 37 ˚C the prepared cultures were incubated for 24 - 72 hours. 0.2 mL of all samples were placed in the holes and 2 mg/mL of Streptomycin was used as standard. The samples were then incubated at 37 ˚C for 24 hours and the inhibition zones were determined in millimeters (Alamzeb et al., 2013).

1H- and 13C-NMR spectra were acquired using deuterated solvents (MeOD, DMSO, and CDCl3) on a Bruker 400-NMR. Various types of NMR procedures (BB, DEPT-90, DEPT-135, HSQC, COSY, and HMBC) were employed for structure identification.

Using glycerol as standard matrix and xenon as gas, mass spectra (JEOL MS Route resolution, and JEOL JMS HX 110 mass spectrometer) were obtained at Husein Ebrahim Jamal research institute of Chemistry, University of Karachi, Pakistan.

20×20 cm aluminum sheets precoated with silica gel (60 F­254) were used. The developed TLC plates were spotted under UV (ultraviolet). Various reagents (Dragent Draft and Cerric Sulphate) were employed for detecting compound nature.

The investigation on B. brevissima roots ethyl acetate fraction (Frac. C) revealed three known (1-3) compounds. The known compounds were identified as β-sitosterol-3-O-β-ᴅ-glucopyranoside (1) (Sabira et al., 2000; Lingamallu et al., 2002), berberine (2) (Shamma et al., 1972; Shamma and Rahimizadeh, 1986), and pakistanine (3) (Shamma and Rahimizadeh, 1986; Shamma et al., 1972). The oil sub-fractions were analyzed through GC-MS and further evaluated for their antimicrobial activities against three bacterial strains.

The GC-MS results of six oil sub-fractions (H-1 - H-6) obtained from B. brevissima were given in Table 1. Linoleic acid was found at the highest concentration (48.34 % and 16.21 %) in H-2 and H-4 sub-fractions respectively, palmitic acid was found in the highest concentration (14.77 % and 6.31 %) in sub-fractions H-1 and H-2 respectively, and oleic acid was found in good amount in sub-fraction H-2 (3.46 %), whereas the results also confirmed the samples as a moderate source of capric acid, myristic acid, margaric acid, pentadecanoic acid, behenic acid, elaidic acid, palmitoleic acid, and tridecanoic acid.

The antibacterial activity of the oil sub-fractions (H-1-H-6) was investigated against Escherichia coli, Staphylococcus epidermis, and Staphylococcus aureus. The results (Table 2) revealed that the H-1 exhibited good activity against E. coli with a zone of inhibition of 10 mm. The H-2 showed noteworthy activity against E. coli (14 mm), while modest activity against S. aureus (10 mm). The H-3 exhibited activity against S. epidermis (11 mm), and E. coli (10 mm) respectively. The H-5 demonstrated the same results against E. coli and S. aureus (12 mm). The H-6 exhibited good results

Table 1: Fatty acids analysis (%) of the oil sub-fractions obtained from the roots of B. brevissima.

Table 2: Anti-microbial activities of the oil sub-fractions obtained from the roots of B. brevissima.

against S. epidermis and E. coli (16 and 14 mm). However, none of the sub-fractions showed as good results as the Streptomycin standard.

Compound 1: Molecular ion peak at m/z 576.43 in FAB-MS, suggested a molecule formula of C12H22O11+, (calcd. C35H50O, 576.43). The 13C-NMR spectrum exposed 35 carbons (6 methyls, 12 methylenes, 14 methines, and 3 quaternary carbons). The aglycone part gives distinctive signals at δC 140.4, 121.1, 76.7, 56.1, 55.4, 49.6, 45.1, 41.8, 39.2, 38.3, 36.8, 36.2, 35.4, 33.3, 31.4, 31.3, 29.2, 28.7, 27.7, 25.4, 23.8, 22.7, 20.5, 19.6, 19.0, 19.0, 18.6, 11.7, and 11.6. The aglycone part was verified to be β-sitosterol by evaluating 13C-NMR data for β-sitosterol (Lingamallu et al., 2002; Sabira et al., 2000).

The 1H-NMR spectra (C5D5N) of the compound 1, confirmed the existence of two peaks at δH 0.64 (3H, s, H-18) and 0.94 (3H, s, H-19) for 2 methyl groups, the signals at δH 0.89 (3H, d, J = 6.4 Hz, H-21), 0.80, (3H, d, J = 7.3 Hz, H-27), and 0.82 (3H, d, J = 7.2 Hz, H-26) indicated 3 doublets methyls. The resonance at δH 0.77 (3H, t, J = 6.9 Hz, H-29) indicated 1 methyl triplets, the peaks at δH 5.29 (1H, m, H-6), indicated the presence of an olefinic proton broad multiplet. The signals at δH 3.45 (1H, m, H-3) indicated the presence of a multiplet. The 1H-NMR of compound 1 exposed the presence of the sugar part, resonated at δH 4.20 (1H, d, J = 7.5 Hz, H-1/), 3.62 (2H, dd, J = 4.5, 10.9 Hz, H-6/), 3.11 (1H, m, H-3/), 3.04 (1H, m, H-4/), 2.88 (1H, m, H-2/), and 3.08 (1H, m, H-5/). The signals in 1H-and 13C-NMR spectra also indicated the sugar part as glucose. From the above spectral data, and by assessment with the literature, the structure of compound 1 was predicted as β-sitosterol-3-O-β-ᴅ-glucopyranoside (Figure 1) (Sabira et al., 2000; Lingamallu et al., 2002).

Compound 2: The molecule formula of compound 2 was suggested to be C20H18N+O4 based on the EI-MS molecular ion peak at m/z 336 (calcd. 336 for C20H18N+O4). The 13C-NMR spectrum displayed twenty signals for 2 methyls, 3 methylenes, 6 methines, and 9 quaternary carbons. The distinctive signals at δC 150.3, 149.9, 148.9, 147.7, 147.7, 146.4, 144.0, 137.5, 130.6, 128.2, 124.5, 120.2, 109.4, 106.5, 103.7, 61.9, 57.7, 55.3, and 28.21 were detected.

The compound 2 1H-NMR spectra resonated at δH 4.10 (3H, s, H-16), and 4.20 (3H, s, H-15) indicated two methyl singlets, the singlet at δH 6.10 (2H, s, H-14) indicated one methylene, the peaks at δH 4.91 (2H, t, J = 6.5 Hz, H-5), and 3.43 (2H, t, J = 6.5 Hz, H-6), showed Two methylene triplets, the peaks observed at δH 7.65 (1H, s, H-4), 6.94 (1H, s, H-1), 8.71 (1H, s, H-13), and 9.75 (1H, s, H-8) confirmed the presence of four methines, and the singlets at δH 8.11 (1H, d, J = 9.6 Hz, H-12), and 8.13 (1H, d, J = 9.6 Hz, H-11) indicated two methine doublets. From the mass and NMR spectra, and the data collected from literature, the compound (2) was elucidated as Berberine (Figure 1) (Shamma et al., 1973; Shamma and Rahimizadeh, 1986).

Compound 3: The molecule formula of compound 3 was suggested to be C37H40N2O6+ based on the EI-MS molecular ion peak at m/z 608 (calcd. 608 for C37H40N2O6). The broadband 13C-NMR spectra indicated thirty-seven peaks containing five methyls, six methylenes, eleven methines, and fifteen quaternary carbons. The peaks at δC 153.64, 147.39, 147.35, 146.90, 143.52, 143.13, 131.60, 128.88, 127.92, 127.91, 124.34, 121.75, 121.35, 118.76, 114.47, 113.99, 111.18, 110.92, 63.98, 61.14, 60.53, 55.98, 54.92, 46.24, 46.04, 42.83, 38.62, 37.54, and 25.60 were observed.

The 1H-NMR spectra of the compound 3, the signals at δH 2.23 (3H, s, H-15, 15/), 3.09 (3H, s, H-16/), 3.12 (3H, s, H-16), and 3.72 (3H, s, H-17), indicated five methyl singlets, the resonance at δH 2.76 (2H, m, H-3, 3/), 2.81 (2H, m, H-4), 2.79 (2H, m, H-4/), 2.90 (2H, m, H-α/), and 2.91 (2H, m, H-α) indicated six methylene multiplets, the peaks at δH 6.94 (1H, s, H-5), 6.96 (1H, s, H-8), 6.78 (1H, s, H-5/), 7.0 (1H, s, H-10/), and 7.08 (1H, s, H-13/) were observed five methine singlets, the signals at δH 7.26 (1H, d, J = 8.1 Hz, H-10, 14), and 7.42 (1H, d, J = 8.1 Hz, H-11, 13) showed four methine doublets and the resonance at δH 4.18 (1H, br. s, H-1), and 4.17 (1H, br. s, H-1/) indicated two methine broad singlets. The obtained mass, NMR spectral data and the data obtained from literature, the structure of compound 3 was elucidated as pakistanine (Figure 1) (Shamma et al., 1972; Shamma et al., 1973).

The fatty acid analysis results of the oils were consistent with the reported ones (Nergiz and Donmez, 2004; Nasri et al., 2005; Koksal et al., 2006). From the current study, it was concluded that the oil sub-fractions of B. brevissima are decent source of fatty acids and are a potential source of antimicrobial agents. Phytochemical investigation on Berberis brevissima Jafri revealed two isoquinoline alkaloids named berberine (2) and pakistanine (3). Research on Berberis species has revealed that alkaloids have anti-microbiocidal properties (Ghoshal et al., 1996). Berberine is the major constituent of the Berberis spp. along with other principal isoquinoline alkaloids (Freile et al., 2003; Ali et al., 2013). Berberine was reported to be the principal antibacterial and antifungal component of the Berberis genus. and is significantly active against S. aureus and Candida spp (Aydemir and Biloglu, 2003; Ali et al., 2013). Berberine has been found effective against many trypanosomes (Ali et al., 2018) and many invertebrate pests (Rattan, 2010).

The current study confirmed the importance of Berberis brevissima Jafri due to the presence of these bioactive components which were already reported from other Berberis species. It is recommended that the current studied plant desires more study for further bioactive components and valued nutraceuticals.

The authors are indebted to the Department of Agricultural Chemistry and Biochemistry, Faculty of Nutrition Sciences, The University of Agriculture Peshawar for providing financial support and necessary laboratory facilities.

Novelty Statement

The Ethyl acetate fraction obtained from the roots of Berberis brevissima Jafri were investigated phytochemically, Fatty acids profile was studied using GC-MS, and antibacterial potential of the obtained oils were also studied.

Zain Ullah: Conducted the research.

Anwar Ali Shad: Designed and supervised the whole research work.

Saqib Ali: Co-supervised the lab work.

The authors have declared no conflict of interest.