Chemical constituents from fruits of Cnestis ferruginea Vahl ex. DC (Connaraceae) and evaluation of their anticholinesterase and antiradical activities

Abstract The phytochemical investigation of the DCM/MeOH (1:1) extract of the fruits of Cnestis ferruginea led to the isolation and characterization of one new quinic acid derivative, ferruginoic acid (1), together with six known compounds 2–7. Compounds 3–7 were reported for the first time from this species. The structures of compounds 1–7 were elucidated on the basis of 1 D and 2 D NMR spectroscopic data, mass spectrometry and by comparison of spectroscopic data with those from the literature. The anticholinesterase (AChE and BChE) activity and DPPH free radical scavenging assay of compounds 1, 3, 4 and 7 were evaluated. Ferruginoic acid (1) exhibited moderate anticholinesterase activity with IC50 value of 36.18 ± 1.78 µg/mL against AChE. Compounds 3, 4 and 7 showed high activity against free radical (DPPH•) scavenging assay (DPPH) with IC50 values 40.09 ± 0.96 µg/mL, 61.70 ± 0.78 µg/mL and 41.87 ± 0.62 µg/mL respectively. These results indicate that C. ferruginea and its constituents could be employed in the management of Alzheimer’s disease. Graphical Abstract


Introduction
Alzheimer's disease (AD) is the most common form of dementia mostly in old people, characterized by low acetylcholine levels and oxidative stress, involving progressive neurodegeneration with formation of amyloid-b deposits in the brain (Tamfu et al. 2020c;Birsan et al. 2021). Worldwide, more than 115 million people will be affected by this disease by 2050 with a majority of them aged above 65 years (Rahman and Choudhary 2015). A proper strategy to overcome AD is through the inhibition of the cholinesterase enzymes, both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which helps to increase acetylcholine levels in the brain and this is necessary for neurotransmission, memory, reasoning, and other cognitive activities (Uddin et al. 2021). Though, cholinesterase inhibitors, such as: rivastigmine, donepezil and galantamine are usually employed as remedy to AD, there is a growing interest in the search of new cholinesterase inhibitors from natural sources due to the draw backs of synthetic ones (Tamfu et al. 2019). Cnestis is a genus, belonging to Connaraceae family and includes about 13 species occurring mostly in the tropics of Africa and Asia (Wiart 2006). Plants of this genus commonly occur as lianes or shrubs and are widely distributed in the tropical regions of South America, Africa and Asia. Scientific studies of these plants reveal interesting biological and chemical potentials, thereby providing new drug leads (Nunes et al. 2020). Some species of this genus were reported to show antioxidant, antimicrobial, antifungal and hypoglycemic activities (Parvez and Rahman 1992;Nijveldt et al. 2001;Adisa et al. 2004Adisa et al. , 2011. Various parts of C. ferruginea including roots, fruits, barks, branches and leaves are used in traditional medicine and have been reported as remedy for diabetes, wounds, diarrhea, periodontitis, headache, snakebites, gum pain, inflammatory conditions, syphilis, dysentery, gonorrhea, scabies, bronchitis, toothache, dysmenorrhea, pains, sinusitis, conjunctivitis and ear problems (Funsho et al. 2013;Catarino et al. 2016;Frazão-Moreira 2016;Ahmed 2017;Lautenschl€ ager et al. 2018;Nunes et al. 2020). These properties of C. ferruginea have attracted much scientific studies and some biological assays have revealed antimicrobial, antioxidant, antiepileptic, antiinflammatory, analgesic, laxative, anti-convulsant, aphrodisiac, hypoglycemic, hepatoprotective, antidepressant properties of the plants (Yakubu and Nurudeen 2012;Owope et al. 2016;Ahmed 2017;Akwasi et al. 2018;Ojo et al. 2019). Phytochemical investigation of some Cnestis species from Africa and Asia report the isolation and characterization of several classes of natural products such as phytosteroids, pentacyclic triterpenoids, flavonoids, coumarin, cinnamoyle (Adisa et al. 2011), and phenolic compounds (Soro et al. 2012).
In this study, C. feruginea was subjected to phytochemical investigation, in search for bioactive secondary metabolites which were in turn evaluated for their anticholinesterase and DPPH radical scavenging activities. The methylene chloride/methanol (1:1) extract of the fruits of C. ferruginea was subjected to column chromatography, leading to the isolation and characterization of one new compound together with six known secondary metabolites. In addition, the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities as well as DPPH radical scavenging activity of compounds 1, 3, 4 and 7 are reported.

Results and discussion
The DCM/MeOH (1:1) extract of the fruits of C. ferruginea was subjected to repeated silica gel column chromatography (CC) which afforded one new quinic acid derivative, together with six known secondary metabolites 1-7 ( Figure 1).
Compounds 1, 3, 4 and 7 were evaluated for their anticholinesterase activity measured by spectrophotometric method on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) as well as their radical scavenging potential on DPPH (2, 2-diphenyl-1-picrylhydrazylhydrazyl) free radical. It was shown previously that the antidepressant activity of C. ferruginea involves enzymatic action notable cholinergic, monoaminergic and L-arginine-nitric oxide pathways (Owope et al. 2016). The antidepressant-like activity of C. ferruginea equally involves antioxidant defense and neuroendocrine systems ). This information suggests that, this plant can possess enzyme inhibitory activity such as anticholinesterase activity. The results obtained (Table S2) show that, ferruginoic acid (1) exhibited moderate activity against anticholinesterase by inhibiting acetylcholinesterase enzyme with IC 50 values of 36.18 ± 1.78 mg/mL compared to the standard Galantamine whose IC 50 value was found to be 5.30 ± 0.16 mg/ mL. Compound 1 exhibited moderate anticholinesterase by inhibiting butyrylcholinesterase enzyme with a percentage inhibition (%) of the enzyme of 34.41 ± 0.90% at 50 mg/mL. These type of low molecular weight terpenoids are major components of essential oils and are known to possess anticholinesterase activities because they are able to interact with AChE and BChE and act as either competitive or noncompetitive inhibitors of cholinesterase due to their lipophilicity and small molecular sizes, which makes them more likely to cross the blood-brain barrier and exert their effect (Alfred et al. 2021;Tamfu et al. 2021). This compound 1 also exhibited moderate activity in the free radical (DPPH ) scavenging assay with a percentage inhibition (%) of 19.39 ± 0.30% at 50 mg/mL. Moreover, compounds 3, 4 and 7 showed good radical (DPPH ) scavenging activity with IC 50 values of 40.09 ± 0.96 mg/mL, 61.70 ± 0.78 mg/mL and 41.87 ± 0.62 mg/mL respectively compared to a-tocopherol with an IC 50 value of 45.31 ± 0.17 mg/mL. The same compounds (3, 4 and 7) exhibited weak activity against anticholinesterase activity determined on AChE and BChE at 50 mg/mL with percentage inhibition values of 10.25 ± 0.55%, 14.37 ± 0.21% and 7.38 ± 0.35% respectively for AChE and 28.03 ± 0.67%, 17.45 ± 0.39% and 20.96 ± 0.83% for BChE respectively. Antiradical and anticholinesterase potential of the compounds is an indication of their possible application as remedy for oxidative stress and Alzheimer's disease(AD). Various medicinal plant extracts and natural compounds have gained special attention due to their specific modes of action and their advantages of low toxicity and high efficiency in the treatment of oxidative stress related ailments and neuroprotective diseases such as AD (Beddiar et al. 2021;Tamfu et al. 2021).

General experimental procedures
High resolution mass spectrum was obtained with a TOF spectrometer (Bruker, South Africa) equipped with an ESI source. The spectrometer was operated in positive and negative modes (mass range: 50-1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 1 ppm deviation using Na Formate as calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200 C. Nitrogen was used as sheath gas (4 L/min). 1 D NMR spectra ( 1 H NMR, 400 MHz; 13 C NMR and DEPT 135; 100 MHz) and 2 D NMR spectra (HSQC, HMBC, COSY and NOESY) were measured on a Bruker Top Spin 3.0 spectrometer equipped with cryoprobe, with TMS as an internal reference. Chemical shifts (d) are expressed in ppm with reference to TMS and coupling constants (J) are given in Hz. Column chromatography (CC) were carried out on silica gel (0.040-0.063 mm). Thin layer chromatography (TLC) were performed on Merck precoated silica gel 60 F 254 aluminum. Spots were visualized under UV light (254 and 366 nm) and by spraying the plates with 10% H 2 SO 4 in EtOH followed by heating at about 110 C.

Plant material
The fruits of C. ferruginea were collected in Kribi locality in the South Region of Cameroon, during the month of December 2013 and was identified by Victor Nana, a botanist of National Herbarium of Cameroon, where a voucher specimen was deposited under the reference 2003/SRF K.

Extraction and isolation
The fruits were chopped, air-dried and pulverized to yield 578.0 g of powder. The resulting powder was macerated three consecutive times in 7.0 L of a mixture of methylene chloride/methanol (1:1) for 72 h at room temperature. The solvent was evaporated under vacuum to afford 83.0 g of methylene chloride/methanol crude extract. A portion of the methylene chloride/methanol extract (80.0 g) was subjected to column chromatography (CC, size: 90 cm, diameter: 5.50 cm) over silica gel (350.0 g) and eluted with a mixture of n-hexane-ethyl acetate and ethyl acetate-methanol gradients polarity. A total 100 fractions of 125 mL each were collected and combined according to TLC profile monitoring to four sub-fractions (F1 to F5). Sub-fraction F2 (3.6 g) was purified employing a step gradient of n-hexane-ethyl acetate and ethyl acetate to yield (3) (3.1 mg) and (2) (19.2 mg). Sub-fraction F3 eluted with a gradient ethyl acetate-methanol yielded (4) (4.2 mg), (5) (2.8 mg), (6) (2.7 mg). Sub-fraction F5 (28.0 g) was subjected to column chromatography on silica gel employing a step gradient of methylene chloride-methanol as eluent, to yield (7) (5.1 mg) and (1) (8.5 mg).

Anticholinesterase activity
The anticholinesterase activity was measured spectrophotometrically by determining acetylcholinesterase and butyrylcholinesterase enzyme inhibition according to the method described by Ellman with minor modifications (Ellman et al. 1961;Tamfu et al. 2020a). Briefly, 130 lL of 100 mM sodium phosphate buffer (pH 8.0), 10 lL of sample solution dissolved in ethanol at various concentrations, and 20 lL of enzyme (AChE or BChE) solution in buffer were mixed and incubated for 15 min at 25 C, followed by 20 lL of 0.5 mM DTNB (5,5 0 -Dithio-bis (2-nitrobenzoic) acid) was added. The reaction was then initiated by addition of 0.71 mM, 20 lL of acetylthiocholine iodide, or 0.2 mM, 20 lL of butyrylthiocholine chloride. The formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride, respectively, was monitored spectrophotometrically using a 96-well microplate reader at a wavelength of 412 nm. The results were expressed as a percentage inhibition (%) of the enzyme at 50 lg/mL concentration of the compounds.

DPPH free radical scavenging assay
The free radical scavenging activity of the compounds were determined by the DPPH (2,2-diphenyl-1-picrylhydrazylhydrazyl) assay as described previously (Tamfu et al. 2020b). In its radical form, DPPH absorbs at 517 nm, but upon reduction by an antioxidant or antiradical species its absorption decreases. Briefly, a 0.1 mmol/L solution of DPPH in methanol was prepared and 4 mL of this solution was added to 1 mL of samples solution in methanol at different concentrations. Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical of an antioxidant was calculated using the following equation: DPPH radical scavenging % ð Þ ¼ AcontrolÀAsample Acontrol X100

Conclusion
Oxidative stress occurs as a result of imbalance between radical species production and antioxidants defense and causes several chronic diseases such as AD. Inhibition of radicals can be a possible solution to these diseases and many plants and their compounds have found applications as natural antioxidants used in the management of oxidative stress related illnesses. In this study, C. ferruginea fruits crude extract and compounds were evaluated for antiradical and anticholinesterase activities. Ferruginoic acid (1) exhibited moderate anticholinesterase activity while compounds 3, 4 and 7 showed good radical (DPPH ) scavenging activity. The biological activities of some isolated compounds as cholinesterase (AChE and BChE) inhibitors, key enzymes involved in hydrolysis of choline leading to AD, partially justify the use of the plant in the treatment of oxidative stress diseases.