Isolation of Pheophytin A and β-amyrin from Newbouldia laevis (P. Beauv) Leaf Extract

. Pulverized leaves samples of Newbouldia laevis were extracted by cold maceration using methanol. The extract was concentrated in vacuo to yield a reddish brown solid of 120.191 g. The crude methanol extract was partitioned into n-hexane 0.1 g, dichloromethane 2.5 g, ethyl acetate 4.6 g, and methanol 10.0 g, fractions via coarse chromatography. Methanol fraction gave the highest yield and was subjected to further purification using repeated column chromatography to yield pure components, namely NLM24 (R f 0.48), EAc:n-hex:MeOH (4:5:1) and NLM19 (R f 0.47), EAc:n-hex:MeOH, respectively. These pure fractions were subjected to 1 H NMR, 13 C, COSY, HSQC and HMBC spectroscopy. Pheophytin A and β-amyrin were proposed as the structures of the isolated compounds. Even though the pure fractions were not used for the analgesic activity, the literature reveals that pheophytin A & β-amyrin are potent analgesics.


INTRODUCTION
Newbouldia laevis (P. Beauv.) is a common plant that is widely used in African traditional medicine [1], and its efficacy against specific health problems such as ulcers, pain, inflammation and microbial infections [2] has been reported and gained wider acceptance. In Nigeria, particularly in the South East, the plant is often used to construct barns for storage of farm produce such as yam, fences around houses and marking of boundaries. In Enugu State, specifically Amede Eha-Amufu and Amankanu, the plant is locally known as ojilishi and is often used to treat wounds.
While studying the analgesic activity of Newbouldia laevis leaf extracts in white whisker albi-no rats, the current research isolated pheophytin A and β-amyrin from Newbouldia laevis.

Materials for Extraction
A solvent distillation machine (PS/1598) is used to distil the solvents, and big glass containers are used for cold maceration.
Precoated microscopic slides were used for spotting; capillary tubes were used for finding; hot air oven ADARSH was used for charring and colour development. Long Big column (60 cm) & 7.0 diameters used for elution of different components; silica gel 60 (70-230 mesh ASTM) used for column chromatography; silica gel 60 (230-400 mesh ASTM) used for flash chromatography.
Spectrophotometer NMR-Bruker AV3-500 MHZ was used for the structure elucidation of isolated pure compounds.
The reagents that were used are listed in Table 1. The leaves were properly washed and air dried. It was further grounded into powder, weighed, and found to be 800 g. It was soaked with distilled methanol, and after two weeks, it was filtered, and the filtrate was refluxed. Thus the solvent was recovered. The crude methanol extract was then kept to air dry. After one week, it was weighed and found to be 120.191 g. It was thus labelled MCNL.
Extraction. After the fresh leaves of N. laevis were collected and identified, it was washed, chopped and dried under shade. The dried leaves were thus pulverized to give 800 g. The 800 g was put in a big glass container, and 100 % of methanol was poured into the container to the brim. The container was thus covered and kept. After two weeks, the sample was filtered, the filtrate was refluxed, and the crude extract was kept to dry.
The residue was resoaked again in 100 % methanol, and one week later, it was filtered, and the filtrate was refluxed; thus, the crude extract was left to air dry. The dried crude extract was weighed to give 120.191 g. It was therefore labelled Newbouldia laevis crude methanol (NLCM).
This method of extraction is called cold maceration. The NLCM obtained was used for bioassay, phytochemical screening and fractionation.
Fractionation. The NLCM 120.191 g was thus partitioned via coarse chromatography to give different fractions as NLH fraction 0.1 g, NLD -2.5 g, NLE -4.6 g, and NLM -10.0 g.
The abbreviations above will be explained later.
Column chromatography. The following procedure was used for the column chromatography: the large column (60 & 7.0 diameter) was hung on a retort stand, and the queue was rinsed with n-hexane. Cotton wool was soaked in the solvent to be used and pushed down the bottom of the column using a steel rod. A mixture of silica gel and poured inside the column. The extract of about (600 mg) was mixed very well with a small silica gel, and the mixture of the crude extract plus the n-hexane and silica gel (slurry) was poured inside the column immediately. About 100 ml of n-hexane was used to wash down the column's sides and fill it up. The solvent system introduced into the column was (n-hexl:EAC 90/10 ml Methanol 100 ml, a more polar solvent, was used to wash off the more polar components remaining in the column. Thin layer chromatography. This technique was used to isolate pure compounds from any fractions collected from column chromatography. Each fraction collected was spotted on a precoated Thin layer Chromatography (TLC) plate with a capillary tube. About four 250 ml beakers were used to develop the spot as it travels from the origin through the solvent front. An Aluminum foil was used to cover the beaker each before the spots travelled through the solvent front. The solvent mixture adopted for a good separation are: 8:2 ml, 7:3 ml (EAc:n-Hex) and 4:5:1 ml (EAc:n-hex:CH 3 OH).
A spot was made on the plate, developed in the solvent front. The dish was brought out from the beaker, 10 % H 2 SO 4 was sprayed on the scale, and charred inside a hot oven at 50 °C for colour visualization. The retention factor for each spot was calculated using the relation (1): At the end of the TLC, similar samples with the same spot were pooled together. Thus NLM24 & NLM19 had single marks each. This means that the fractions are pure; therefore, they were packaged and sent for spectral analysis.
Spectroscopic analysis. Spectroscopy studies the interactions between particles such as electrons, protons and ions, as well as their interaction with other particles as a function of their collision energy.
To be more precise, spectroscopy is the study of absorption and emission of light and other radiation by matter, as related to the dependence of these processes on the wavelength of the radiation. For the research, the following are the instrument, samples obtained and the laboratory where the experiment was done.

RESULTS AND DISCUSSION
The extracts NLM, NLE, and NLDCM obtained were used to conduct chemical analysis viz: phytochemical screening, column chromatography, thin layer chromatography, and NMR spectroscopy. Column chromatography was done using about 10.0 g of methanol extract. The vials label, volume of the solvent mixture used for elution (ml) and colour of fractions when visualized with the naked eye are shown in Table 4 above.
When the solvent mixture was changed to Hexane/Ethyl acetate (60/40), ten fractions (5 ml each) of NLD we collected and another five fractions (5 ml each) of NLD were collected when the solvent mixture was changed to Hexane/Ethyl acetate (50/50). Their corresponding colours are shown in the table above. Still, on the solvent mixture of Hexane/Ethyl acetate (50/50) and changing the solvent mixture (40/60, 30/70 and 20/80), 30 fraction (5 ml each) of NLE were collected. The colours corresponding to each of them are seen in Table 4 above.
Changing the solvent mixture further to Hexane/Ethyl acetate (10/90, 0/100) and finally washing down with 100 ml methanol, 30 fractions (5 ml each) of NLM were collected, and the colour of each particle can be seen in the Spectral analysis results show in Table 5. The Nitrogen resonances were not seen. They were on the opposing side.
The signal at C-13 1 (σ190.4) is the carbonyl of the cleaved E-ring.
Signals at 104.20 ppm, 97.90 ppm and 93.63 ppm were observed in the negative phase corresponding to the olefinic methine (=CH) carbons of σ104.2 (C-10), σ97.9 (C-5) and σ93.6 (C-20), which indicates a porphyrin moiety. Also, the signals at 51.90 ppm and 50.40 ppm observed in the positive phase corresponded to σ51.9 (C-17) and σ50.4 (C-18) methine carbons of the porphyrin moiety, while the signals at 61.58 ppm (COOCH , P 1 ) corresponds to the oxymethylene carbon which confirmed the esterification of the porphyrin ring by phytyl group. More so, the signal at 117.78(C P 2 )is characteristic of the olefinic carbon of the phytol group. The triplet at 77 ppm in the 13 C spectrum was due to the solvent (Deuterated chloroform) signal.
Signals were observed at 3.74 ppm (3H, d 18 1 ) and 1.72 ppm (3H, t8 2 ), resulting from four methyls and one ethyl group bonded to the pyrrole ring of the porphyrin unit. Also, the 1 H-1 H correlation signals at 4.48 ppm (2H, m-P 1 ) and 5.26 ppm (1H, t-P 2 ) were assigned to the ester and olefinic protons of the phytyl group and correlated in the 13 C Spectrum of NLM24 with carbon signals at 61.58 ppm (2H, m-P 1 ) and 117.94 ppm (=CH-P 2 ). This confirms the esterification of the porphyrin moiety at C-17 3 by phytol.
The methine protons at 4.16 ppm (H-17) and 4.48 ppm (H-18) also correlated to C-17 through 3 J and 2 J coupling, respectively. The methyl protons at 1.92 ppm (H-18 1 ) connected to C-17 at 51.90 ppm and C-18 at 50.40 ppm through 3 J and 2 J collar with the methine carbon (C-18).
Hence, the oxymethylene protons at 4.48 ppm (H-P 1 ) correlated to the (olefinic) carbon at 117.94 ppm (C-P 2 ), establishing the presence of the phytyl group in the structure (NLM24).
Isolation of pheophytin A from Newbouldial aevis leaf is hereby reported for the first time. Pheophytin A is an Mg-free analogue of chlorophyll formed by replacing the Mg 2+ in the chlorophyll molecules with (2H). NLM24 is brownish. It is practically insoluble in water but soluble in ethanol, diethyl ether, chloroalkanes and hydrocarbons.
It is from the above information as well as comparison with the literature data that led to the characterization of NLM19 as β-amyrin. The results were in good agreement with previous reports from [10]. Isolation of β-amyrin from Newbouldia laevis leaf is hereby reported for the first time. The amyrins are three closely related natural chemical compounds of triterpenes. Amyrins can exist as α-amyrin, β-amyrin and δ-amyrin [11].
The isolated compound "β-amyrin" from NLM19 is a pentacyclic triterpenoid, an oleanane substituted at the three beta-position by a hydroxyl group with a double bond between carbon positions 12 and 13.
β-amyrin was first isolated in 1968 by corey and gross. β-amyrin possesses a white colour and is primarily white solid upon isolation. The compound "β-amyrin" from NLM19 is soluble in ethanol and dimethyl formamide (DMF).