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A New Biflavonoid Glycoside from the Leaves of Ziziphus mucronata Willd. (Rhamnaceae)

The use of natural products especially plants for healing is as ancient and universal as medicine itself because they have been an integral part of the ancient traditional medicine systems such as the Chinese, Ayurvedic and Egyptian systems of medicine. Natural products are known to possess several phytochemicals responsible for their pharmacological activities or acts as the precursor for the synthesis of novel drugs. Even now, continuous traditions of natural product therapy exist throughout the third world, especially in the occident, where numerous minerals, animal substances and plants are still in common use. One of such plants is Ziziphus mucronata of the rhamnaceae family. It is commonly known as buffalo thorn and is a spiny shrub or small to medium-sized tree that grows up to 20 m high with a spreading canopy. The leaves are used in African traditional medicine for the treatment of diarrhoea, tumour, cough, sores, ear inflammation, asthma, syphilis, gonorrhoea, measles, fever and psychiatric disorder. Phytochemical screening of the methanol extract of the leaves had revealed the presence of saponins, flavonoids, tannins, triterpenes and steroids; catechins and triterpenoids were previously isolated from the leaves of the plant. The ethnomedicinal use of the leaves in the treatment of pain and inflammation was recently validated scientifically. Despite its varied medicinal potentials, there is paucity of information on the isolation and characterization of bioactive compounds from this plant; only the isolation of cyclopeptides have been reported in the scientific database. In continuation of our work aimed at isolating the principles responsible for the observed diverse biological activities, we present herein, the isolation


Introduction
The use of natural products especially plants for healing is as ancient and universal as medicine itself because they have been an integral part of the ancient traditional medicine systems such as the Chinese, Ayurvedic and Egyptian systems of medicine. 1 Natural products are known to possess several phytochemicals responsible for their pharmacological activities or acts as the precursor for the synthesis of novel drugs. 2 Even now, continuous traditions of natural product therapy exist throughout the third world, especially in the occident, where numerous minerals, animal substances and plants are still in common use. One of such plants is Ziziphus mucronata of the rhamnaceae family. It is commonly known as buffalo thorn and is a spiny shrub or small to medium-sized tree that grows up to 20 m high with a spreading canopy. 3 The leaves are used in African traditional medicine for the treatment of diarrhoea, tumour, cough, sores, ear inflammation, asthma, syphilis, gonorrhoea, measles, fever and psychiatric disorder. 3 Phytochemical screening of the methanol extract of the leaves had revealed the presence of saponins, flavonoids, tannins, triterpenes and steroids; catechins 4 and triterpenoids were previously isolated from the leaves of the plant. The ethnomedicinal use of the leaves in the treatment of pain and inflammation was recently validated scientifically. 5 Despite its varied medicinal potentials, there is paucity of information on the isolation and characterization of bioactive compounds from this plant; only the isolation of cyclopeptides have been reported in the scientific database. 6 In continuation of our work aimed at isolating the principles responsible for the observed diverse biological activities, we present herein, the isolation

General experimental procedures
The solvents used were of high quality (analytical grade) and include: methanol, n -hexane, chloroform, ethyl acetate and n-butanol purchased from Sigma Co. USA; silica gel 60-120 µm (Qualikems, India) was used for column chromatography, sephadex LH-20 (GE Healthcare) was used for purification of isolated compound, thin layer chromatography (TLC) was carried out on aluminum-backed Kieselgel 60 F254 TLC plate (Merck no. 5554, Darmstadt, Germany) and a Gallenkhamp electro thermal melting point apparatus was used to determine the melting point of isolated compound. The UV was recorded on Thermo scientific biomate 6 UV-Visible spectrophotometer; the IR was recorded on Agilent Technologies Cary 6030 Fourier Transform Infrared Spectrophotometer; 1 H __ NMR (500 MHz, MeOD) and 13 C __ NMR (125 MHz, MeOD) spectra were recorded on a Bruker AVANCE-500 spectrophotometer (Japan). The chemical shift values (δ) were reported in parts per million (ppm) relative to internal standard TMS and coupling constants (J values) were given in Hertz.

Collection and identification of the plant material
The leaves of Z. mucronata were collected from Kudingi village, Giwa Local Government Area of Kaduna State, Nigeria in 2015. It was authenticated by taxonomist Musa Muhammad in the Herbarium Unit, Department of Biological Sciences, Ahmadu Bello University, Zaria by comparison with the existing herbarium specimen, voucher number 900328.

Preparation of the extract
The leaves of the plant were dried under shade at room temperature until a constant weight was obtained and size-reduced manually using mortar and pestle. About 1 kg of the pulverized leaf material was extracted with methanol by cold maceration for 72 hours with occasional shaking and concentrated in vacuo using rotary evaporator at 40 o C. This yielded a dark green gummy (134 g) crude extract; 100 g of the extract was suspended in 500 mL of distilled water and filtered using Whatman No. 1 filter paper.
The water-soluble portion was partitioned successively with 400 mL each of ethyl acetate and n-butanol to obtain ethyl acetate fraction (EAF) and n-butanol fraction (nBF), respectively.
Column Chromatography of n-butanol fraction About 4 g of the n-butanol soluble fraction of the extract was adsorbed onto about 6 g of silica gel and mounted over a glass column (75 cm x 3.5 cm) packed with 200 g silica gel and chromatographed. The column was eluted continuously with ethyl acetate 100 % followed by gradient mixtures of ethyl acetate and methanol; the progress of separation was monitored using TLC with solvent system: ethyl acetate/chloroform/methanol/water: 15:8:4:1; a total of 68 collections of 50 mL aliquots each, was made and similar collections were pooled together based on their TLC profile to obtain thirteen major fractions coded A-M. Fraction F (12.2 mg) obtained from the solvent gradient mixture, ethyl acetate/methanol 90:10, containing three major spots was subjected to further purification using sephadex-LH20, eluting with methanol. The progress of separation was monitored on TLC plate. Repeated gel filtration afforded 4.8 mg yellow crystalline solid compound that was subsequently coded as 'S1'. The compound gave a single homogenous spot with two solvent systems of different polarity, ethyl acetate/chloroform/methanol/water; 15:8:4:1 and 15:4:4:1 indicating its purity. The isolated compound, S1, was subjected to physical and some chemical tests as well as spectral analysis to elucidate its chemical structure.

Results and Discussion
The pulverized leaf material (1 kg) yielded 13.4 % w/w crude methanol extract; 7.5 % ethyl acetate fraction (EAF) and 12 % n-butanol fraction (nBF) were respectively obtained from the water-soluble portion of the extract. The compound S1 revealed an uncorrected melting point range of 243-245°C and was found to be soluble in methanol and insoluble in n-hexane. It gave a Prussian blue colour with freshly prepared ferric chloride solution indicating the presence of phenolic nucleus 7 ; the compound also produced a red colour with concentrated hydrochloric acid in the presence of magnesium chips (Shinoda test). This is indicative of a flavonoid nucleus. 7

Spectral Analysis
The UV spectrum of figure 1 showed two absorption bands within the UV region (λmax 226 nm and 257 nm) and two bands within the visible region (λmax 346 nm and 363 nm) in methanol. These shoulder bands are characteristic of compounds with a highly conjugated system. 8 The Infrared spectrophotometer spectrum of figure 1 showed two important peaks at 1697 cm -1 and 3447 cm -1 on a KBr disc due to carbonyl and hydroxyl functional groups, respectively.  figure  1 suggest that the compound, S1, has a biflavonoid nucleus. Ten of the methine signals clustered around δH (3.22 -3.85) corresponding to carbon signals δC (62.0 -78.5) suggest the presence of two sugar residues. The two anomeric proton signals at [δH 5.15 (1H, d, J = 8.0 Hz, H-1*) and 5.24 (1H, d, J = 7.5 Hz, H-1**)] are typical of glucose. 9 The high coupling constants observed (8.0 Hz and 7.5 Hz) are due to diaxial coupling with H-2 proton of each of the residue and confirmed the configuration of both sugars as β-D glucosyl. 9 The proton noise decoupled 13 C-NMR disclosed 42 significantly overlapping peaks. The presence of two carbonyl carbon peaks at δC 179.48 and 179.44 further confirmed the dimeric nature of S1. The DEPT experiment revealed the presence of 20 methine carbon resonances; the two methylene carbon signals at δC 62.6 (C-6*) and δC 62.0 (C-6**) also indicated the two sugar moieties as glucose. Moreover, the C-3 resonances observed in the downfield region (δC 135.7 and δC 135.8) relative to C-10 (δC 105.0) (the same chemical environment) further confirmed that the glycosylation is at C-3 position. 10 The 13 C __ NMR also showed ten oxygen bearing quaternary carbons. Six carbons have hydroxyl groups attached and two are linked to pyranone oxygen. This suggested that the remaining two should be involved in interflavonoid ether linkage. Thus, S1 must be a biflavonoid with either 4'-4'' or 3'-4'' ether linkage. 11 The downfield resonance of C-4'' compared to C-4' suggested a 4'-4'' linkage. The linkage was further confirmed by analyzing the heteronuclear multiple bond correlation (HMBC) spectral data (see below). The 1 H -1 H COSY spectra of S1 also showed important cross peaks between δH 3.82 and δH 3.47 and the two sugar moieties at δH 5.15 and δH 5.24, in addition to the ortho and meta correlations observed in the benzene rings. Other important COSY correlations occurred between carbinol protons attached to adjacent carbons in the two sugar residues (Figure 2). The heteronuclear multiple quantum coherence experiment (HMQC) facilitated the attachment of all protons to their respective carbons ( Table  1). The unambiguous assignment of the carbons and the placement of the sugar moieties were facilitated using the long-range correlation experiments (HMBC) ( Table 2). In the spectrum, a common J3 correlation between protons at δH 6.19 and 6.38 to carbons at δC 95.1 and 100.4, respectively confirmed their assignment to rings A of both flavonoid residues. Also, a J2 correlation between protons at δH 6.88 to carbons at δC 123.0 and 123.3 and a J3 correlation to carbon at δC 146.0 confirmed their assignments to ring B. A J3 correlation between anomeric protons at δH 5.15 and 5.24 to the quaternary carbons at δC 135.7 and 135.8 confirmed that each of the sugar moiety is linked to the individual flavonoid aglycone through C-3 carbon, thereby confirming the biflavonoid diglycoside to be derived from a flavonol nucleus. Other correlations confirmed by the HMBC are those of the sugar residue. Based on the foregoing spectral analysis, S1 was confirmed to be a biflavonoid and with the aid of 2D NMR correlations, the structure of S1 was proposed to be bis (quercetin 3-O-β-D-glucopyranoside) ( Figure 1). C-O-C linked biflavonoids are those in which two monomeric units may be of the same or different structural types joined to each other through an ether linkage. 12 Unlike C-C linked biflavonoids, C-O-C linked biflavonoids have restricted distribution in the plant kingdom. 11 They are classified into various groups due to the nature of the interflavonyl linkage between them and have been used as chemotaxonomic markers for different families of plant e.g. Ochna flavones first isolated from the Ochnaceae family. 13 Some of the reported pharmacological activities of biflavonoids include: inhibition of histamine release from mast cells and inhibition of lymphocyte proliferation, suggesting the anti-inflammatory/anti-allergic potential of biflavonoids.

Conclusion
The isolation of this biflavonoid from Z. mucronata seems to support some folkloric use of the plant in African traditional medicine, in the management of inflammatory conditions. Anti-inflammatory studies and the possible mechanism of action of compound S1 are in progress in our laboratory.