Saponins from the Pericarp of Albizia lebbeck (Benth) L. and Their Biological Screening on Laboratory Animals

Aim: Albizia lebbeck L. is widely used to treat many diseases. Saponins are one of the major constituents that are found in the tissues of the tree. The purpose of this study was aimed at the phytochemical and biological investigation of the saponins of the pericarp. Methodology: 50% ethanol extract of the dried powdered pericarp of A. lebbeck was fractionated with different organic solvents and examinated for their oxidative stress radical, anticonvulsant and analgesic effects. The highly potent fraction was subjected to chromatographic and spectroscopic analysis of its saponins. Results: The n-butanol fraction expressed the best results regarding the biological activities. Chromatographic and spectroscopic analysis of this fraction revealed the identification of three oleanane-type saponins. Original Research Article Eid and Sokkar; JPRI, 33(28B): 183-192, 2021; Article no.JPRI.68285 184 Conclusion: The present study constituted the first phytochemical analysis of the bioactive saponins content in the pericarp of A. lebbeck.

In continuation to our studies on A. lebbeck tree [9,10], the biological activity was performed on the 50% ethanol extract and its fractions of the pericarp. A detailed phytochemical investigation of the highly potent saponins in n-butanol fraction pod was presented here; structures were based mainly on NMR and LC-MSn (n=2).

Solvents
Acetonitrile (HPLC grade, Merck), deionized H 2 O was treated with pure aqua RC655. Solvents and chemical reagents were of analytical grade (BDH).

Standards and Reagents
Vitamin E, glacial acetic acid and carbamazepine were kindly supplied from the NRC, Giza, Egypt.

Extraction
50% ethanol was used to extract air-dried and defatted (using petroleum ether) powdered pericarp (700 g) of Albizia lebbeck L. to give 71.5 g (10.1%) dry residue. A part of the residue was dissolved in water and portioned in succession with organic solvents. The dry yield was 15.7 g chloroform (CFP), 4.4 g ethyl acetate (EAFP) and 4.1 g butanol (BFP).

Sample for the Biology
The test samples (2 g, each) were dissolved separately in distilled water (20%, w/v) with tween 80 (few drops).

Animals
Male albino rats (130-150 g) of Sprague Dawley strain and albino mice (25-30 g) were employed in this study. They were fed a standard laboratory diet, and kept under standardized conditions with water given ad lib.

Antioxidant Activity
Glutathione in the blood was evaluated according to Beutler et al., (1963) [12]. Adult male albino rats (130-150 g) were split into seven groups (6 rats/group). Group 1 (control), while diabetes milletus was induced in the remaining groups using a single intraperitoneal injection of 150 mg alloxan /kg b. wt, followed by fasting overnight [13]. Group 2 of diabetic rats were left untreated, group 3 received vitamin E (7.5 mg /kg b. wt.), and the groups (4-7) received orally 50% alcoholic extract, CFP, EAFP and BFP respectively, (100 mg / kg b. wt. each). The glutathione determination in blood was carried out after one week.

Analgesic Activity
The acetic acid-induced writhing test was used to assess the analgesic activity. Five groups (6 mice/group). Group 1 (control) received 1ml saline orally, the groups (2-5) of mice received 50% alcoholic extract, CFP, EAFP and BFP respectively, orally one hour later (100 mg/kg b. wt., each); 0.6% acetic acid was administered intraperitoneally (0.2 ml/mice) in the groups. Mice were then individually placed in a clear plastic observational chamber, and the number of writhes/30 min was counted for each mouse.

Anticonvulsant Activity
This activity was carried out, using Frequently Pulse Shock Current apparatus. Male albino mice (25-30 g) were split into six groups (6 mice/group). Group 1 (control) and was given 1ml saline orally, the groups (2-5) received the test samples. All doses were taken orally one hour before the experiment (100 mg/kg b. wt., each). Group 6 received carbamazepine. The stimulus duration was 0.2 second, the end-point was tonic hide limb extension, the maximum electric shock needed to emit a cry was determined.

Phytochemical Work
BFP (2 g/200 mlmethanol) was dissolved in 5 times of its volume acetone where a precipitate was formed, isolated by centrifuging and purified by repeated dissolution and precipitationto give 250 mg dry powder. A portion of the powder (200 mg) was dissolved in 50% aqueous acetonitrile (20 ml) and injected in preparative HPLC, at 25°C. Fractions relevant to each peak were collected and concentrated to afford compounds: 1(40 mg), 2 (38 mg) and 3(35 mg). NMR of compounds 1-3 (Tables 1& 2). Compounds 1-3 (8-10 mg, each) were separately refluxed with 2N HCl in 50% methanol (10 ml) for 2 h. The aglycone was extracted with chloroform and subjected to TLC (S1) and spectral analysis. The sugars were liberated after neutralization with Ag 2 CO 3 and identified by comparison with authentics using GC [14].

Biological Analysis
Toxicity study revealed that oral administration ofthe test samples in dose up to 1.3 g/ kg. Blood glutathione ( Fig. 1) was restored in diabetic animals following oral administration of the test samples.
The BFP showed a significant antioxidant activity (3.57%). It may be attributed to the presence of compounds can produce stable free radicals and thus inhibit the autooxidation of co-existing substances acting as biological antioxidants [14]. The 50% alcoholic extract induced significant analgesic effect (Fig.  2), as it increased numbers of abdominal constrictions of mice by 61.09% followed by the BFP that increased the constrictions by 47.28%.
Peripheral analgesic agents are known to specifically produce a constriction response of abdomen due to acetic acid. Prostaglandins are believed to underlie this response [15]. The 50% extract and BFP resulted in antinociceptive activity indicating that they may contain analgesic components which could be involved in modulation of the prostaglandin pathway.
50% alcoholic extract as well as the BFP and EAFP induced significant anticonvulsant effects (Fig. 3) through the increase of the % protection by: 241.66%,179.16% and 145.83, respectively. This activity may be attributed to fractions that resulted in an elevated level of serotonin and gamma-aminobutyric acid (GABA). In particular, these fractions exhibited a central nervous system depressant action [16].

Phytochemical Investigation
The significant results of the BFP encouraged the authors for its phytochemical investigation. Previous work discussed saponins isolated from different tissues of A. lebbeck of echinocystic acid [3], acacic acid lactone [1] and oleanolic acid [2] aglycones. In the present study, the saponins were isolated by preparative HPLC.  [17].
Gas chromatography ofthe sugars alongside with authentics revealed the glucose, rhamnose, arabinose, xylose and quinovose.
Compound 2( Table 1) is an analogue to 1 with the difference of the presence of the terminal glup" in the oligosaccharide "a" at C-3. 1 H-NMR showed its anomeric proton at δ H 5.38 (d, J=7.8Hz) which correlated in the HSQC spectrum with 13 C-NMR spectrum of C-1 at δ C 101.7. The HMBC suggested the attachment between H-glup"-1 / C-glup-2 at δH 5.38 / δC 83.4. NMR spectrum of 3 (Tables 1 & 2) showed a structure close to 1 and 2; their C-arap-2 showed more downfield shifts (δ C 81.4 and 81.1, respectively) for the sugar substitutions than in 3 which appeared at δ C 72.6 indicating its terminal position. The structure of "a" fragment is superimposable to that of the oligosaccharide chain attached to C-1 of kinmoonosides isolated from Acacia concinna [20].    Assignments of the sugars based on HSQC-TOCSY and 13 C NMR.

CONCLUSION
This study constitutes an investigation of the sponins in the pericarp of A. lebbeck. They proved to have a valuable antioxidant, analgesic and anticonvulsant activities; through examination of the n-butanol fraction, containing them and having a complex structure of an acacic acid aglycone with oligosaccharide substitutions at C-3 and C-28 and by acyl groups at C-21. They are different from the Albiziahexosides previously isolated from leaves and the by-product of its degradation reaction albiziatrioside A [2], Albiziasaponins isolated from the bark [1] or echinocystic acid glycosides isolated from the seeds [3] of A. lebbeck. To our knowledge, this is the first investigation of saponins identification from the pericarp of A. lebbeck.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The animal study was approved by the Research Ethics Committee, Faculty of Pharmacy, King Abdulaziz University, approval number PH-1442-74.