with n-hexane–CHCl (6.0:4.0) was rechromatographed and eluted with n-hexane–CHCl (7.0:3.0) to furnish β-sitosterol (5)
3
3
(15 mg) and oleanolic acid (6) (10 mg). On increasing the polarity with n-hexane–CHCl (5.0:5.0), gallic acid (7) (12 mg) was
3
isolated. Fraction EtA eluted with n-hexane–CHCl (5.0:5.0) was again chromatographed and eluted with mixtures of
2
3
n-hexane and CHCl in increasing order of polarity. The fraction obtained with n-hexane–CHCl (3.0:7.0) provided berberisinol
3
3
(1) (35 mg). Fraction EA obtained from n-hexane–CHCl (2.0:8.0) was rechromatographed and eluted with CHCl –MeOH
3
3
3
(4.5:5.5, 4.0:6.0, and 2.0:8.0) to afford berberine (3) (12 mg), 8-oxoberberine (4) (10 mg), and palmatine (2) (14 mg), respectively.
Berberisinol (1) was obtained as a yellow gummy solid, which gave a violet coloration with FeCl for a phenol. UV
3
(MeOH, λ , nm) (log ε): 220 (2.28), 288 (3.31). The molecular formula was established as C H O through HR-EI-MS
max
26 28 5
+
–1
1
13
showing an [M] peak at m/z 420.1937 (calcd 420.1929). IR (KBr, ν , cm ): 3380, 1690, 1660, and 1615. H, C NMR,
max
see Table 1.
Acid Hydrolysis. A solution of berberisinol (4 mg) in MeOH (2 mL) containing 1 N HCl (2 mL) was refluxed for
4 h, concentrated under reduced pressure, and diluted with H O (4 mL). It was extracted with CH Cl , and the organic phase
2
2
2
was extracted with dilute KOH, washed with water, dried, and freed of solvent under reduced pressure to afford a colorless
+
liquid (bp 187°C), characterized as 2-ethylhexanol (M peak at m/z 130, superimposable IR). The aqueous layer was acidified
with dilute HCl and extracted with EtOAc. The residue recovered from the organic phase was found to be an inseparable
mixture of products.
Bioassays. The antifungal activity was performed on different pathogens. The growth inhibitory potential of
compound 1 and standard drug (both at the concentration of 100 μg/mL of Sabouraud glucose agar) was tested against
different strains, A. niger (ATCC-10549), F. solani (ATCC-11712), C. glabrata (ATCC-90030), A. flavus (ATCC-32611),
C. albicans (ATCC-2091), and M. canis (ATCC-11622) according to the previously reported protocol [21], and the results are
illustrated in Table 2.
Similarly, compound 1 and standard drugs (each at a concentration of 100 μg/mL of nutrient agar) were tested for
antibacterial potential. Antibacterial bioassay was carried out on six strains, including Gram-positive bacteria S. pyogenes
(ATCC-29213), S. aureus (ATCC-25923), and B. subtilis (ATCC-6051), Gram-negative bacteria P. aeruginosa (ATCC-27853),
E. coli (ATCC-25922), and K. pneumoniae (ATCC-700603) according to the literature [19]. The results are presented in Table 3.
The antioxidant activity of compound 1 (IC 52.5 0.4 μg/10 μL) was determined by the DPPH free radical scavenging
50
activity method [22]. Butylhydroxyanisole (IC 42.5 0.6 μg/10 μL) was employed as standard.
50
ACKNOWLEDGMENT
We are grateful to Prof. Dr. Abdul Malik of HEJ Research Institute of Chemistry, University of Karachi for his help
in structural elucidation of isolated compounds.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
T. Khan, I. A. Khan, and A. Rehman, J. Med. Plants, 2 (6), 16 (2014).
I. Khan, S. Najeebullah, M. Ali, and Z. K. Shinwari, Trop. J. Pharm. Res., 15, 2047 (2016).
R. Rao, T. Husain, B. Dutt, and A. Garg, Rheedea-Kerala, 8, 1 (1998).
A. Perveen and M. Qaiser, Pak. J. Bot., 42, 1 (2010).
F. Aqil and I. Ahmad, Meth. Find Exp. Clin. Pharmacol., 29, 79 (2007).
R. Tareen and S. Qadir, Pak. J. Bot., 23, 90 (1991).
N. Baloch, S. Nabi, M. Yasser, and A. Kahraman, J. Phytopharmacol., 4, 282 (2013).
T. Bibi, M.Ahmad, R. B. Tareen, N. M. Jabeen, R. Rehman, S. U. Sultana, S. Zafar, and M. Yaseen, J. Ethnopharmacol.,
157, 79 (2014).
9.
10.
11.
12.
M. Bi, Iqbal, J. Khan, T. Shahwani, N. A. Razzaque, G. Baloch, and M. Y. Baloch, J. Sci. Techlol., 2306, 185 (2016).
A. Karimov, M. Levkovich, N. Abdullaev, and R. Shakirov, Chem. Nat. Compd., 29, 361 (1993).
I. M. Tadzhibaev, I. Zatorskaya, K. Lutfullin, and T. Shakirov, Chem. Nat. Compd., 10, 42 (1974).
N. Kovganko and Z. N. Kashkan, Chem. Nat. Compd., 30, 533 (1994).
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